ISFG 2019 Program

F THE INTER O NA SS T E IO R N G A N L O C

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T E N E S G O C C SI IET N Y FOR FORE ISFG PRAGUE

THE 28th CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS

9 – 13th SEPTEMBER 2019 PRAGUE CONGRESS CENTRE, THE CZECH REPUBLIC www.isfg2019.org ABSTRACT BOOK

Content

DETAILED SCIENTIFIC PROGRAMME 2 ORAL 11 POSTERS 81 FORENSIC GENETICS AND GENOMICS OF HUMAN AND NON‑HUMAN BIOLOGICAL SAMPLES 82 POPULATION GENETICS AND FORENSIC DNA DATABASES 268 DNA TYPING METHODOLOGIES AND STRATEGIES 406 FORENSIC MATHEMATICS AND STATISTICS 573 DNA PHENOTYPING, PHARMACOGENETICS, AND EPIGENETICS 612 NEW POLYMORPHISMS OF FORENSIC INTEREST 667 STANDARDS, QUALITY CONTROL, ACCREDITATION, AND ETHICS 699 AUTHOR INDEX 723

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 1 Detailed Scientific Programme

2 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Tuesday 10 th September 2019, Forum Hall 18:30 OPENING CEREMONY 19:00 OPENING CEREMONY & SCIENTIFIC PRIZE LECTURE FORENSIC APPEARANCE PREDICTION FROM DNA: A JOURNEY THROUGH 10 YEARS OF SCIENTIFIC CONTRIBUTIONS Kayer, Manfred 20:00 WELCOME RECEPTION (FOYER)

Wednesday 11th September, Forum Hall

Session 1 Chairs: Walther Parson, Marie Korabečná

08:30 KEYNOTE LECTURE THE WHITE ELEPHANT IN THE FIELD – WHAT MEANS “POPULATION“ IN FORENSIC GENETICS? Roewer, Lutz 09:15 DEVELOPMENT AND OPTIMIZATION OF THE VISAGE PROTOTYPE TOOLS FOR BIO-GEOGRAPHIC ANCESTRY AND APPEARANCE TRAITS INFERENCE USING TARGETED MPS Xavier, Catarina 09:30 APPROACHES TO EXPLAIN AND POTENTIALLY PREDICT THE COMPLEX ARCHITECTURE OF THE HUMAN FACE Walsh, Susan 09:45 INTRODUCTION OF A PREDICTIVE DNA TEST FOR THE OCCURRENCE OF FRECKLES Branicki, Wojciech 10:00 COFFEE BREAK (FOYER) & POSTER SESSION (SOUTH HALL 2)

Session 2 Chairs: Walther Parson, Marie Korabečná

11:00 FORENSIC DNA PHENOTYPING: A SERVICE PROVIDER TRIAL Raymond, Jennifer 11:12 COMPARISON OF CE- AND MPS-BASED ANALYSES OF FORENSIC MARKERS WITH SINGLE CELL AFTER WHOLE GENOME AMPLIFICATION Chen, Man

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 3 11:24 PRESENTATION OF THE HUMAN PIGMENTATION (HUPI) AMPLISEQ CUSTOM PANEL Meyer, Olivia Strunge 11:36 PREDICTIVE DNA ANALYSIS OF HUMAN HEAD HAIR GREYING USING WHOLE-EXOME AND TARGETED NGS DATA EXAMINED WITH DEEP LEARNING METHODS Pośpiech, Ewelina 11:48 A COMPARISON OF DNA METHYLATION TECHNOLOGIES AND PERFORMANCE OF AGE PREDICTION MODELS Freire-Aradas, Ana 12:00 A COMING OF AGE TALE Aliferi, Anastasia 12:12 IN SEARCH OF CENTRAL- AND EASTERN EUROPEAN- SPECIFIC ANCESTRY INFORMATIVE MARKERS Woźniak, Marcin 12:30 COMPANY SYMPOSIUM BY THERMO FISHER SCIENTIFIC 13:00 LUNCH 13:30 COMPANY SYMPOSIUM BY QIAGEN

Session 3 Chairs: John Butler, Manfred Kayser

14:30 KEYNOTE LECTURE EXPLAINING BAYESIAN INFERENCE PRINCIPLES NONVERBALLY: HOW TO HELP NON-MATHEMATICIANS UNDERSTANDING THE WEIGHT OF EVIDENCE Šimková, Halina 15:15 HOW TO AVOID DRIVING DNA CASEWORKERS CRAZY: CASESOLVER, AN EXPERT SYSTEM TO INVESTIGATE COMPLEX CRIME SCENES Prieto, Lourdes 15:30 COMPARISON OF CE AND MPS BASED ANALYSIS FOR THE PROBABILISTIC INTERPRETATION OF MIXED STR PROFILES Benschop, Corina 15:45 USING GENETIC COMPLEXITY TO SOLVE FORENSIC COMPLEXITY: A NEW CLASS OF COMPLEX HYPERVARIABLE STR MARKERS FOR DECONVOLUTION OF COMPLEX DNA MIXTURES Ralf, Arwin 16:00 COFFEE BREAK (FOYER)

4 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Session 4 Chairs: John Butler, Manfred Kayser

16:30 THE FIRST MPS-STR BASED CONVICTION IN A CRIMINAL CASE? De Knijff, Peter 16:42 ENHANCING STR SEQUENCE ALLELE REPRESENTATION FOR PROBABILISTIC GENOTYPING Just, Rebecca 16:54 A MASSIVELY PARALLEL SEQUENCING ASSAY OF MICROHAPLOTYPES FOR MIXTURE DECONVOLUTION Oldoni, Fabio 17:06 A TOP-DOWN APPROACH TO MIXTURE EVALUATION Slooten, Klaas 17:18 FROM REFERENCE TO MIXTURE TO MIXTURE TO MIXTURE AND BEYOND Kruijver, Maarten 17:30 EXPLORING DNA INTERPRETATION SOFTWARE USING THE PROVEDIT DATASET Riman, Sarah 17:42 ARE REPORTED LIKELIHOOD RATIOS WELL CALIBRATED? Hannig, Jan 18:00 WORKING GROUPS MEETINGS

Thursday 12 th September 2019, Forum Hall

Session 5 Chairs: Mechthild Prinz, Jiří Drábek

8:30 KEYNOTE LECTURE FORENSIC GENETICS AND DTC GENOMICS: FRIEND OR FOE? Erlich, Yaniv 9:15 DEVELOPING PRIORITIES FOR DISCUSSION AND OVERSIGHT OF THE RAPIDLY EVOLVING FIELD OF GENETIC GENEALOGY Phillips, Christopher 9:30 THE EFFECTIVENESS OF FORENSIC GENEALOGY TECHNIQUES IN THE UNITED KINGDOM – AN EXPERIMENTAL ASSESSMENT Thomson, Jim

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 5 9:45 FORENSIC GENEALOGY – PERFORMANCE OF DENSE SNP DATA TO TRACE DISTANT RELATIVES Kling, Daniel 10:00 COFFEE BREAK (FOYER) & POSTER SESSION (SOUTH HALL 2)

Session 6 Chairs: Mechthild Prinz, Jiří Drábek

11:00 AN INTERNATIONAL CONSIDERATION OF A STANDARDS-BASED APPROACH TO FORENSIC GENETIC GENEALOGY Scudder, Nathan 11:12 WHOLE GENOME SEQUENCING OF HUMAN REMAINS TO ENABLE GENEALOGY DNA DATABASE SEARCHES – A CASE REPORT Tillmar, Andreas 11:24 ETHICAL, SOCIAL AND LEGAL ISSUES OF FAMILIAL SEARCHING: NEW AND OLD DEBATES Granja, Rafaela 11:36 THE GENETIC IDENTIFICATION OF DEAD MIGRANTS IN THE MEDITERRANEAN SEA: THE LAMPEDUSA 2013 SHIPWRECK Bertoglio, Barbara 11:48 RE-EVALUATION OF DNA BASED IDENTIFICATION RESULTS OF VICTIMS OF A TERRORIST ATTACK 25 YEARS LATER Corach, Daniel 12:00 NEW ISO STANDARDS FOR FORENSICS: DNA “FREE” CONSUMABLES AND THE FORENSIC PROCESS Bastisch, Ingo 12:12 REFLECTIONS AND EXAMPLES OF PROBLEMATIC REPORTING IN DNA CASES: THE NEED FOR ACCREDITED FORMATS AND CERTIFIED REPORTING COMPETENCE Hicks, Tacha 12:30 COMPANY SYMPOSIUM BY PROMEGA 13:20 LUNCH

Session 7 Chairs: Peter Schneider, Titia Sijen

14:30 KEYNOTE LECTURE DNA TRANSFER: ASPECTS RELEVANT TO FORENSIC INVESTIGATIONS van Oorschot, Roland

6 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS 15:15 TRANSFER, PERSISTENCE AND RECOVERY OF EPITHELIAL CELLS ON THE SKIN IN DIRECT AND SECONDARY TRANSFER SCENARIOS Fonneløp, Ane Elida 15:30 MODELLING DNA TRANSFERS IN COMPLEX SCENARIOS Taylor, Duncan 15:45 ASSIGNING FORENSIC BODY FLUIDS TO DNA DONORS IN MIXED SAMPLES BY TARGETED RNA/DNA DEEP SEQUENCING OF CODING REGION SNPS USING ION TORRENT TECHNOLOGY Ballantyne, John 16:00 COFFEE BREAK (FOYER)

Session 8 Chairs: Peter Schneider, Titia Sijen

16:30 VISUALISING DNA TRANSFER: LATENT DNA DETECTION USING DIAMOND DYE Champion, Jessica 16:42 IN AND OUT OF TOUCH: RELATIVE ACCUMULATION OF CELLULAR AND ACELLULAR “TOUCH DNA” FROM ENDOGENOUS AND EXOGENOUS SOURCES ON HANDS OVER TIME Burrill, Julie 16:54 CHARACTERIZATION OF TISSUE-SPECIFIC BIOMARKERS WITH THE EXPRESSION OF CIRCRNAS IN FORENSICALLY RELEVANT BODY FLUIDS Yang, Qinrui 17:06 BODY FLUID IDENTIFICATION USING MRNA – BETTER, FASTER, CHEAPER – WHAT METHOD IS BEST OR SHOULD A COMBINATION OF TECHNIQUES BE USED? Harbison, Sallyann 17:18 PREDICTING THE ORIGIN OF FORENSICALLY RELEVANT BIOLOGICAL MATERIAL USING A MACHINE LEARNING APPROACH Iacob, Diana 17:30 DEVELOPMENT OF A MIRNA BODY FLUID PREDICTION SYSTEM USING PROBABILISTIC APPROACHES Li, Zhilong 17:42 PROTEOMIC GENOTYPING: USING MASS SPECTROMETRY TO INFER SNP GENOTYPES IN A FORENSIC CONTEXT Parker, Glendon 18:00 GENERAL ASSEMBLY

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 7 Friday 13th October 2019, Forum Hall

Session 9 Chairs: Leonor Gusmão, Halina Šimková

8:30 KEYNOTE LECTURE BACKGROUND SELECTION AND BIASED GENE CONVERSION AFFECT MORE THAN 95 % OF THE HUMAN GENOME AND BIAS DEMOGRAPHIC INFERENCES Pouyet, Fanny 9:15 Y-PROFILE EVIDENCE: CLOSE PATERNAL RELATIVES AND MIXTURES Andersen, Mikkel Meyer 9:30 INFERENCE OF ADMIXED ANCESTRY WITH ANCESTRY INFORMATIVE MARKERS Tvedebrink, Torben 9:45 THE IMPACT OF IGNORING INBREEDING IN KINSHIP EVALUATIONS Kjelgaard Brustad, Hilde 10:00 COFFEE BREAK (FOYER) & POSTER SESSION (SOUTH HALL 2)

Session 10 Chairs: Leonor Gusmão, Halina Šimková

11:00 STR SEQUENCE NOMENCLATURE: PROGRESS REPORT FROM THE STRAND WORKING GROUP Gettings, Katherine 11:12 THE STRIDER REPORT ON QUALITY CONTROL OF AUTOSOMAL STR DATASETS – THE GOOD, THE NOT SO GOOD AND THE UGLY Bodner, Martin 11:24 ADVANCING MITOCHONDRIAL GENOME DATA INTERPRETATION IN MISSING PERSONS CASEWORK Marshall, Charla 11:36 CSY? A PANEL-BASED MPS APPROACH INCLUDING 12,523 Y-CHROMOSOME POLYMORPHISMS Claerhout, Sofie 11:48 ANALYSIS OF RECOMBINATION AND MUTATION EVENTS FOR 12 X-CHR STR LOCI: A COLLABORATIVE FAMILY STUDY OF THE ITALIAN SPEAKING WORKING GROUP GE.F.I. Bini, Carla

8 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS 12:00 GENETIC PEOPLING OF PAKISTAN AND THE IMPACT OF HISTORICAL MIGRATIONS, ETHNIC CULTURES AND THE PRACTICE OF ENDOGAMY ON THE FORENSIC MATCH PROBABILITIES Anwar, Ijaz 12:12 THE SCOPE AND LIMITATIONS OF THE LIKELIHOOD RATIO METHOD APPLIED TO STR DATA FOR DETERMINING GENETIC KINSHIP IN ANCIENT OR ISOLATED HUMAN POPULATIONS Zvenigorosky, Vincent 12:30 LUNCH

Session 11 Chairs: Adrian Linacre, Eugenia D’Amato

14:00 KEYNOTE LECTURE USING MICROBIOME TOOLS TO ESTIMATE THE POSTMORTEM INTERVAL OF HUMAN REMAINS Metcalf, Jessica 14:45 ENVIRONMENTAL DNA TO ASSIST FORENSIC INVESTIGATIONS, COUNTER‑TERRORISM, FRAUDULENT MEDICINES AND DRUG SEIZURES Young, Jennifer 15:00 TAXONOMY-INDEPENDENT DEEP LEARNING MICROBIOME APPROACH FOR ACCURATE CLASSIFICATION OF FORENSICALLY RELEVANT HUMAN BIOMATERIALS USING TARGETED MPS Díez López, Celia 15:15 PERFORMANCE OF ENVIRONMENTAL DNA METABARCODING IN SOIL TRACE MATCHING AND PROVENANCING Frøslev, Tobias Guldberg 15:30 COFFEE BREAK (FOYER)

Session 12 Chairs: Adrian Linacre, Eugenia D’Amato

16:00 COLLABORATION ACROSS BORDERS TO IMPLEMENT A EUROPEAN DATABASE FOR CANINE STR IDENTIFICATION MARKERS Giangasparo, Federica 16:12 MULTI-LOCUS DNA METABARCODING FOR AUTHENTICATION OF HIGHLY PROCESSED MEAT PRODUCTS COLLECTED IN SOUTH AFRICA Pietroni, Carlotta

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 9 16:24 SPECIES IDENTIFICATION USING MASSIVELY PARALLEL SEQUENCING – DETECTING MULTIPLE SPECIES IN MIXED SOURCES Dellamico, Barbara 16:36 SPECIES IDENTIFICATION IN ROUTINE CASEWORK SAMPLES USING THE SPINDEL KIT Pereira, Filipe 16:48 WHOLE-GENOME SEQUENCING OF NEISSERIA GONORRHOEAE IN A FORENSIC TRANSMISSION CASE Gonzalez-Candelas, Fernando 17:00 WHOLE TRANSCRIPTOME ANALYSIS OF AGED BIOLOGICAL CRIME SCENE TRACES Salzmann, Andrea 17:12 PREVALENCE OF DNA IN VEHICLES: OPTIMIZING SAMPLING STRATEGY AND ACTIVITY LEVEL EVALUATION OF DNA TYPING RESULTS Kokshoorn, Bas 19:30 CLOSING CEREMONY & CONGRESS DINNER (MUNICIPAL HOUSE – SMETANA HALL)

10 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Oral

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 11 SCIENTIFIC PRIZE LECTURE

O001 - FORENSIC APPEARANCE PREDICTION FROM DNA: A JOURNEY THROUGH 10 YEARS OF SCIENTIFIC CONTRIBUTIONS Manfred Kayser1

1 Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, the Netherlands

With great honour and appreciation I received The ISFG Biennial Scientific Prize 2017 for outstanding contributions regarding my “work related to forensic DNA phenotyping and haploid markers”. On the occasion of the ISFG Scientific Prize lecture, I will take the audience on a 10 years journey of the scientific contributions my department, together with our collaborators, made to the field of forensic DNA phenotyping, particularly regarding the genetic basis and genetic prediction of human appearance for forensic applications, which begun in 2009. Within the last decade, we substantially increased the genetic knowledge of human appearance by finding new genes for eye colour, hair colour, skin colour, skin tanning, eyebrow colour, eyebrow thickness, body height, head hair shape, sagging eyelids, facial morphology, facial pigmented spots, facial wrinkles, and facial age. When the unveiled genetic appearance information was large enough, we developed and validated statistical models for predicting eye colour, hair colour, skin colour, eyebrow colour, tall stature, head hair shape, and male pattern baldness from genotype data. When the accuracies achieved by the appearance prediction models were high enough, we developed and forensically validated lab tools and made online available statistical tools for predicting eye colour, hair colour, and skin colour from DNA, which we (and others) applied in forensic casework (and anthropological studies). I am grateful to the colleagues from my department, from other departments within Erasmus MC, and the many international collaborators without whom these scientific achievements would not have been possible. To allow focus, my additionally awarded work on haploid markers will not be covered by this ISFG Scientific Prize lecture.

12 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS KEYNOTE LECTURE

O002 - THE WHITE ELEPHANT IN THE FIELD – WHAT MEANS “POPULATION“ IN FORENSIC GENETICS? Roewer Lutz1

1 Charité Universitätsmedizin, Institute of Legal Medicine and Forensic Sciences, Berlin, Germany

To study genetic variants, a forensic scientist has to leave his laboratory and step into the field in order to collect samples from a group of people. A group of people – this is the easiest answer what a human population is. A more precise definition would be a “group of people of interest”, implying that the researcher has a certain knowledge about the properties of the polymorphic system under study. For example, if the Y chromosome is studied, the scientist would probably avoid the sampling of female persons. Of course outreach is limited, hence the scientist has to develop a strategy to collect a “group of people of interest which is representative” for the larger population. In the context of forensic DNA typing this could be the whole population or the population from which the suspect comes, the “suspect population”. That in turn implies that the researcher has enough knowledge on the structure of the population, its history, its current composition. Knowing this the scientist could do his collection according to the “subpopulation structure”. However, what if self-perception, political categorization (census) and knowledge-based assignment of a putative proband differs? And another complication lurks just around the corner: to make meaningful inferences genetic traits need to be studied in a random sample, i.e. as far as possible unrelated individuals. How can the scientist sample the unrelated without knowing the relationship structure in the target population, the family systems and the social organization? Assuming this issue is solved and the scientist has collected random samples in the sub-populations representing the population as a whole, the next problem is naming his population sample, a final irritation. Science-based name systems rarely coincide with population names which are used by law enforcement or other authorities acting in the name of the state. The first and final hurdle is therefore: how to communicate to the audience what a population is. This talk is based on own experiences in the field but owes much to the many colleagues who tirelessly collect and intrepidly name population samples for the YHRD.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 13 O003 - DEVELOPMENT AND OPTIMIZATION OF THE VISAGE PROTOTYPE TOOLS FOR BIO-GEOGRAPHIC ANCESTRY AND APPEARANCE TRAITS INFERENCE USING TARGETED MPS Catarina Xavier1, María de la Puente1,2, Antonia Heidegger1, Harald Niederstätter1, Carsten Hohoff3, Christopher Phillips2, Manfred Kayser4, Walther Parson on behalf of the VISAGE Consortium1,5

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Forensic Genetics Unit- Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Institut für Forensiche Genetik, GmbH, Münster, Germany 4 Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, Netherlands 5 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

DNA intelligence is increasing in interest and applications within the forensic genetics community. The ability of providing investigative leads based on appearance, biogeographic ancestry (BGA) and age estimation, particularly in cases where standard DNA profiling methods are non-informative, makes these new markers and tools valuable contributions to future routine casework. Thus, there is an increasing interest in such DNA markers, tools, and statistical models to allow for user-friendly, cost-effective usage in forensic casework provided legal allowance. In this context, the VISible Attributes through GEnomics (VISAGE) Consortium was founded to promote, develop, forensically validate and implement new prototype tools for appearance, BGA and chronological age inference from trace DNA (http://www.visage-h2020.eu/). The VISAGE Project comprises two phases, each producing working and forensically validated tools for Ap- pearance, Ancestry (A&A) and Age inference from DNA with two levels of detail. Here we present the results of two years of VISAGE describing the development, optimization, and forensic validation of the A&A tools. They were designed using state of the art DNA markers and employ targeted massively parallel sequencing using AmpliSeq design and technology (Thermo Fisher Sci- entific). The results from the forensic validation of the basic tool (153 SNPs) and the preliminary results from the enhanced tool (418 SNPs and 22 microhaplotypes) show both prototypes to be robust and highly sensitive, proving their suitability for further forensic implementation testing. This study received support from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 740580 within the framework of the VISAGE Project and Consortium.

14 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O004 - APPROACHES TO EXPLAIN AND POTENTIALLY PREDICT THE COMPLEX ARCHITECTURE OF THE HUMAN FACE Ryan J. Eller1, Julie D. White2, Karlijne Indencleef3, Sahin Naqvi4, Jasmien Roosenboom5, Myoung Keun Lee5, Jaaved Mohammed6, Stephen Richmond7, Mary Marazita5, Eleanor Feingold8, Greet Hens9, Joanna Wysocka6, John R. Shaffer8, Seth M. Weinberg5, Mark D. Shriver2, Peter Claes3, Susan Walsh1

1 Indiana University Purdue University Indianapolis, Department of Biology, Indianapolis IN, USA 2 The Pennsylvania State University, Department of Anthropology, University Park PA, USA 3 KU Leuven, Department of Electrical Engineering, Leuven, Belgium 4 Massachusetts Institute of Technology, Department of Biology, Cambridge MA, USA 5 University of Pittsburgh, Department of Oral Biology, Pittsburgh PA, USA 6 Stanford University, Department of Chemical and Systems Biology, Stanford CA, USA 7 Cardiff University, Applied Clinical Research and Public Health, Cardiff, United Kingdom 8 University of Pittsburgh, Department of Human Genetics, Pittsburgh PA, USA 9 KU leuven, Department of Otorhinolaryngology, Leuven, Belgium

The human face is a highly complex trait with many genetic signatures. Advances in computational and graphical approaches to facial segmentation using 3D imagery now allow a data-driven approach to phenotyping facial shape (Claes 2018). With the ability to generate parameters that include all aspects of facial variation using this shape space, more fundamental multivariate statistical approaches can be used to determine how groups of genetic variants are effectively working together to explain facial variation via a structural relationship. Structural equation modeling (SEM) is a form of causal modeling that attempts to define the cause-effect relationship between observed and unobserved variables. Using a list of genetic variants (n=203) significantly associated with face shape, found in a recent genome-wide association study of 8,246 US and UK individuals that utilized this phenotyping segmentation approach, we assessed if and how these variants may collectively explain the variation observed in each segment of the face. Of the 63 facial segments, 57 of the models converged on a solution successfully and output a ranked list of variants, thereby identifying groups of particular loci that affect certain parts of the face. In addition, the SEM approach allowed for the detection of epistatic interactions within these groups of variants. Four segments were influenced by pairs of SNPs, with significant masking effects, providing additional proof of the complex genetic architecture of the face. We therefore attempt to provide insight into potential biological relationships that may be at play during the formation of the face from a statistical modeling point of view. Such examinations provide great potential for the ability to predict the human face in the coming years.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 15 O005 - INTRODUCTION OF A PREDICTIVE DNA TEST FOR THE OCCURRENCE OF FRECKLES Magdalena Kukla-Bartoszek1, Ewelina Pośpiech2, Anna Woźniak3, Michał Boroń3, Joanna Karłowska‑Pik4, Magdalena Zubańska5, Agnieszka Bronikowska6, Tomasz Grzybowski7, Rafał Płoski8, Magdalena Spólnicka3, Wojciech Branicki2,3

1 Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Poland 2 Malopolska Centre of Biotechnology of the Jagiellonian University, Laboratory of Human Genome Variation Research, Krakow, Poland 3 Central Forensic Laboratory of the Police, Depertment of Biology, Warsaw, Poland 4 Nicolaus Copernicus University, Faculty of Mathematics and Computer Science, Torun, Poland 5 Police Academy, Faculty of Security and Legal Sciences, Institute for Security Sciences, Szczytno, Poland 6 Collegium Medicum of the Jagiellonian University, Department of Dermatology, Kraków, Poland 7 Collegium Medicum of the Nicolaus Copernicus University, Department of Forensic Medicine, Division of Molecular and Forensic Genetics, Bydgoszcz, Poland 8 Warsaw Medical University, Department of Medical Genetics, Warsaw, Poland

Ephelides are small hyperpigmented spots observed on skin surface mainly in European and Asian populations. Predictive DNA analysis of freckles may have practical value in the field of forensic phenotyping. Because of higher prevalence in people with light pigmentation it is expected that predisposition to freckles formation may be partially controlled by pigmentation genes. In this study we examined 113 candidate DNA variants previously associated with human pigmentation traits investigating their impact on freckles occurrence in a cohort of 960 individuals. Significant association with freckles was revealed for 19 DNA variants and sex. Furthermore, we identified a synergistic interaction between rs1042602 in TYR and rs9894429 in NPLOC4 explaining 2.9 % of the total variation in freckle occurrence that had positive impact on prediction accuracy. We propose two alternative regression models for prediction of freckles occurrence. A simplified binomial 12-variable model predicts the presence of ephelides with AUC=0.75. A multinomial 14-variable model predicts the number of freckles based on classification of individuals to one of the three categories, non-freckled, medium freckled or heavily freckled. The two extreme categories, non-freckled and heavily freckled were predicted with moderately high accuracy of AUC=0.75 and 0.79, respectively. Prediction accuracy of the intermediate category was lower with AUC=0.66. The total variation in freckles occurrence explained by the variants included in the multinomial model equalled 33 %. In conclusion, we propose to the forensic community two predictive DNA tests for the occurrence of freckles, which extend the list of physical appearance traits predicted in the field of forensic DNA phenotyping.

16 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O006 - FORENSIC DNA PHENOTYPING: A SERVICE PROVIDER TRIAL Jennifer Raymond1, Runa Daniel2, Lauren Atwood1, Alison Sears3, Michael Bell1

1 New South Wales Police Force, Forensic Evidence & Technical Services Command, Parramatta, Australia 2 Victoria Police, Forensic Services Department, Macleod, Australia 3 NSW Health Pathology, Forensic and Analytical Science Service, Lidcombe, Australia

Forensic intelligence is a crucial component of criminal investigations, with investigators asking more from forensic science to assist in casework resolution and support. In the absence of a DNA database profile match, information regarding the donor’s appearance and ancestry can be used to generate or prioritise a suspect pool, where the investigation would otherwise stall. Forensic DNA phenotyping (FDP) is the prediction of a person’s externally visible characteristics (hair and eye colour) and biogeographic ancestry from their DNA. FDP has been in existence for a number of years but has yet to become a routine element of forensic examinations in many jurisdictions. Prior to implementing this technology in criminal or coronial casework, the New South Wales Police Force commissioned an assessment of a number of FDP service providers. Six commercial service providers were selected, comprising three academic institutions and three private en- terprises. Ten donors with a variety of phenotypic traits and ancestral backgrounds volunteered blood and saliva samples to be distributed anonymously to each of the providers. The providers were primarily assessed according to the accuracy of their predictions against the donor’s appearance and self-declared ancestry. However, the quality and comprehensibility of their reports, turn-around times, cost, and ease of use were also evaluated. This presentation will outline the results of the project and highlight considerations for organisations seeking to implement such a service into their investigative toolkit.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 17 O007 - COMPARISON OF CE – AND MPS-BASED ANALYSES OF FORENSIC MARKERS WITH SINGLE CELL AFTER WHOLE GENOME AMPLIFICATION Man Chen1, Jingjing Zhang2, Xu Liu3, Jing Zhao4, Zhiyong Liu1, Qingwei Fan5, Feng Cheng5, Tong Chen1, Jiangwei Yan5

1 University of Chinese Academy of Sciences, Beijing Institute of Genomics, Beijing, China 2 Beijing Huayan Judicial Authentication Institute, Beijing Huayan Judicial Authentication Institute, Beijing, China 3 Beijing Center for Physical and Chemical Analysis, Judicial Identification Center of Beijing Center for Physical and Chemical Analysis, Beijing, China 4 CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Beijing, China 5 Shanxi Medical University, School of Forensic Medicine, Taiyuan, China

Whole genome amplification (WGA) allowed multiple genetic analyses with low template DNA even a single cell. WGA could increase the amount of input-DNA from pg up to µg-level. How- ever, studies comparing the performance of forensic markers with single cell after WGA on both capillary electrophoresis (CE) and massively parallel sequencing (MPS) platforms remain absent. In this study, one female cultured B-lymphoblastoid and one karyocyte from male venous blood cell lines were segregated into one, two, three and five cells, respectively. Including the reference samples with bulk cells, all samples were generated WGA of multiple displacement amplification (MDA) strategy in triple, and genotyped on CE and MPS platforms, respectively. Al- lele balance, stutter ratio, accuracy, repeatability and concordance of STR markers were used to evaluate the genotyping performance on both platforms. Additionally, sequence coverage ratio (SCR) and SNP genotypes were also evaluated for the sequence information generated from the MPS. Heterozygotic loci showed high allele balance with the whole average allele balance ratio bigger than 0.62 on CE and 0.70 of MPS platforms of the cultured cell samples, as the venous blood cell samples was 0.79 and 0.76, correspondingly. Stutter ratio of every source and number cell line samples were very closed, ranged from 5.3 % to 7 % of autosomal STR and around 10 % of Y chromosomal STR on CE platform. Average stutter, allele, sequence-based and length-based noise ratios were 6.6 %, 88 %, 4.7 % and 0.7 % of the single venous blood cell sample, respectively. SNPs were showed high consistency and intralocus balance too. Our study indicated that WGA of MDA may become an alternative method to increase the success rate of forensic profiling from low copy number DNA.

18 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O008 - PRESENTATION OF THE HUMAN PIGMENTATION (HUPI) AMPLISEQ CUSTOM PANEL Olivia Strunge Meyer1, Jeppe Dyrberg Andersen1, Claus Børsting1

1 Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V’s vej 11- 2100 Copenhagen, Denmark

Externally visible traits include pigmentation of skin, eyes, and hair. These traits are among the most apparent characteristics of human diversity and useful to describe an (unknown) individual. Human pigmentation is highly heritable and only moderately influenced by environmental factors. The high heritability is essential for accurate prediction of a trait by DNA typing. Many genetic variants with association to normal pigmentary variation have been identified, and assays for pigmentary trait prediction have been developed. The assays use different chemistries for genotyping. Examples include Single Base Extension (detected by CE or MALDI-TOF MS) and TaqMan, which have limited multiplexing abilities. Here, we present the Human Pigmentation (HuPi) AmpliSeq custom panel designed for the Ion S5™ System. The panel was designed as one large multiplex PCR using the Ion AmpliSeq Designer v7.0.6 (Thermo Fisher Scientific). The PCR amplifies 184 SNPs and InDels known to influence normal pigmentary variation of the eyes, skin, and hair in 49 genes including OCA2, MC1R, SLC24A4, SLC45A2, TYR, and TYRP1. The 41 SNPs from the HIrisPlex-S assay were also included. The HuPi panel consists of 164 amplicons. The average amplicon size is 118 bp (70–137 bp), which makes the panel applicable for forensic casework. Of the 184 targets, 16 were eliminated in downstream analyses due to no or low coverage (<30x). Twelve of the 16 variants were in complete or strong LD with other variants in the HuPi panel. An initial sensitivity study resulted in full, concordant profiles using 100 pg total input DNA. Performance of the HuPi panel, i.e. locus balance, heterozygote balance, and noise levels will be presented.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 19 O009 - PREDICTIVE DNA ANALYSIS OF HUMAN HEAD HAIR GREYING USING WHOLE-EXOME AND TARGETED NGS DATA EXAMINED WITH DEEP LEARNING METHODS Ewelina Pośpiech1, Magdalena Kukla-Bartoszek2, Anna Woźniak3, Michał Boroń3, Joanna Karłowska‑Pik4, Piotr Zieliński5, Magdalena Zubańska6, Agnieszka Bronikowska7, Tomasz Grzybowski8, Rafał Płoski9, Magdalena Spólnicka3, Wojciech Branicki1,3

1 Jagiellonian University in Krakow, Malopolska Centre of Biotechnology, Krakow, Poland 2 Jagiellonian University in Krakow, Faculty of Biochemistry Biophysics and Biotechnology, Krakow, Poland 3 Central Forensic Laboratory of the Police, Biology Department, Warsaw, Poland 4 Nicolaus Copernicus University, Faculty of Mathematics and Computer Science, Torun, Poland 5 Jagiellonian University in Krakow, Institute of Environmental Sciences, Krakow, Poland 6 Police Academy in Szczytno, Institute for Security Sciences Faculty of Security and Legal Sciences, Szczytno, Poland 7 Collegium Medicum of the Jagiellonian University, Department of Dermatology, Krakow, Poland 8 Collegium Medicum of the Nicolaus Copernicus University, Department of Forensic Medicine Division of Molecular and Forensic Genetics, Bydgoszcz, Poland 9 Warsaw Medical University, Department of Medical Genetics, Warsaw, Poland

Aging is inherently connected with changes in human appearance. Understanding the mechanisms of differences in the pace of changes in human appearance may have practical meaning for the forensic DNA intelligence. Hair greying is caused by a progressive loss of pigment from the growing hair shaft. In addition to age, sex- and ancestry-specific patterns of hair greying progression are also observed but little is known about the genetic control of this process. This study aimed to evaluate the potential of multiple SNPs to predict hair greying status. The discovery stage involved whole-exome sequencing (WES) of 149 samples and subsequent selection of 34 candidate SNPs. Additionally, 344 SNPs previously associated with pigmentation, hair shape and hair loss were also selected for analysis. The whole set of SNPs was genotyped in two Ion AmpliSeqTM NGS panels and evaluated in the remaining set of 849 subjects. Predictors were pre-selected using minimum redundancy maximum relevance feature selection approach (mRMRe) and a neural network method was applied for modelling. The final model included age, sex and 15 SNPs including 3 WES-identified SNPs and 6 SNPs previously linked with hair loss. The final model allowed prediction of hair greying with AUC=0.89 for no greying, 0.84 for slightly greying and 0.92 for significant greying categories. Our study shows that age alone explains 45.5 % of the variation observed in hair greying while the identified DNA variants are characterized by small effect sizes and explain 6.9 % of the total variability. Our study presents the prototype model for hair greying and proves that appearance prediction pipeline needs to contain sex, age and ancestry information in order to make DNA phenotyping a reliable approach in forensic investigations.

20 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O010 - A COMPARISON OF DNA METHYLATION TECHNOLOGIES AND PERFORMANCE OF AGE PREDICTION MODELS Ana Freire-Aradas1, Ewelina Pośpiech2, Anastasia Aliferi3, Lorena Girón‑Santamaría1, Ana Mosquera Miguel1, Adrián Ambroa1, Christopher Phillips1, María de los Ángeles Casares de Cal4, Antonio Gómez-Tato4, Jose Álvarez-Dios4, David Ballard3, Denise Syndercombe Court3, Wojciech Branicki2, María Victoria Lareu1

1 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic Genetics Unit, Santiago de Compostela, Spain 2 Jagiellonian University, Malopolska Centre of Biotechnology, Kraków, Poland 3 King’s College London, Faculty of Life Sciences and Medicine, Department of Analytical, Environmental and Forensic Sciences, London, United Kingdom 4 University of Santiago de Compostela, Faculty of Mathematics, Santiago de Compostela, Spain

Individual age estimation can be applied to criminal, legal and anthropological investigations. DNA methylation has been established as the biomarker of choice for age prediction; since it was observed that specific CpG positions in the genome show systematic changes during an individual’s lifetime, with progressive increases or decreases in methylation levels. Subsequently, several forensic age prediction models have been reported, providing average age prediction error ranges of ±3–4 years, using a broad spectrum of technologies and underlying statistical analyses. DNA methylation is not a categorical but a quantitative trait; therefore the detection platform used plays a pivotal role, since quantitative and semi-quantitative technologies could potentially provide differences in detected DNA methylation levels. In the present study, we analyzed common blood-based DNA controls ranging from 18 to 99 years old using four different technologies: EpiTYPER, Pyrosequencing, MiSeq and SNaP- shotTM. The DNA methylation levels detected with each system were compared. A restricted three CpG-site age prediction model was rebuilt for each system and all samples had their ages predicted. We describe comparisons of the performance of the detection systems and predictive models applied to the panel of common controls of known age.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 21 O011 - A COMING OF AGE TALE Anastasia Aliferi1, Sudha Sundaram1, Ana Freire-Aradas2, David Ballard1, Christopher Phillips2, María Victoria Lareu2, Denise Syndercombe Court1

1 King’s College London, King's Forensics, Department of Analytical Environmental and Forensic Sciences, London, United Kingdom 2 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic Genetics Unit, Santiago de Compostela, Spain

The field of forensic DNA intelligence has been advancing rapidly in recent years, with DNA methylation-based age prediction being one of the main focus points of current research. How- ever, while numerous studies have shown promising results using a variety of different markers, the forensic community has yet to reach a consensus on a marker set that would provide maximum prediction accuracy and sensitivity in a forensic context. As part of this work, 49 recent studies referencing over 36,000 unique age-correlated CpG sites in whole blood were investigated and methylation data for approximately 8,500 individuals were collected from publicly available datasets for the ~6,300 most prominent markers. Extensive statistical analysis on this data revealed a final set of less than 20 CpGs that appear to be superior in both their correlation with chronological age and their potential for sensitivity in terms of starting DNA inputs. Following the development of this panel, further methylation analysis for these markers was performed on a new set of ~200 whole blood samples collected from individuals aged between 11 and 93 years. The data obtained from this analysis was then used to establish the accuracy for this panel while using DNA inputs as low as 1ng. This analysis was performed using the Illumina MiSeq FGx benchtop instrument coupled with a machine learning approach. This systematic approach to marker selection, designed to maximize both prediction accuracy and assay sensitivity, offers the first comprehensive insight into the future of this type of predictive tool in forensic casework.

22 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O012 - IN SEARCH OF CENTRAL- AND EASTERN EUROPEAN- SPECIFIC ANCESTRY INFORMATIVE MARKERS Marcin Woźniak1, Urszula Rogalla‑Ładniak1, Renata Zbieć-Piekarska2, Anna Woźniak2, Boroń Michał2, Boris A. Malyarchuk3, Miroslava V Derenko3, Nataša Kovačević-Grujičić4, Milena Stevanović5, Krzysztof Rębała6, Rafał Płoski7, Ewelina Pośpiech8, Magdalena Kukla-Bartoszek9, Magdalena Zubańska10, Wojciech Branicki8, Magdalena Spólnicka2, Tomasz Grzybowski1

1 Collegium Medicum of the Nicolaus Copernicus University, Department of Forensic Medicine, Division of Molecular and Forensic Genetics-, Bydgoszcz, Poland 2 Central Forensic Laboratory of the Police, Laboratory of Forensic Biology, Warsaw, Poland 3 Russian Academy of Sciences, Institute of Biological Problems of the North, Magadan, Russian Federation 4 University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia 5 University of Belgrade, Faculty of Biology, Belgrade, Serbia 6 Medical University of Gdańsk, Chair of Forensic Medicine, Gdańsk, Poland 7 Warsaw Medical University, Department of Medical Genetics, Warsaw, Poland 8 Jagiellonian University, Malopolska Centre of Biotechnology, Krakow, Poland 9 Jagiellonian University, Faculty of Biochemistr, Biophysics and Biotechnology, Krakow, Poland 10 Police Academy, Institute for Security Sciences- Faculty of Security and Legal Sciences, Szczytno, Poland

Genetic diversity of Central and Eastern European populations is underrepresented in genomic databases and projects. It is assumed that the genetic components present in this part of Eu- rope do not differ significantly from other parts of the continent, thus rendering the search for biologically and medically important genetic variants redundant. However, a number of studies point to the existence of appreciable differences in allele and haplotype frequencies between Central and East Europe (CE Europe) and the rest of the continent. Such differences may lead to detection of ancestry-informative markers (AIMs) specific to the region. In the search for AIMs specific for CE Europe we have performed whole-exome sequencing of about 200 samples of Slavic origin (Poles, Serbs and Russians) and identified SNPs whose allele frequencies were significantly different between those populations as well as from other Euro- pean populations. Additionally, we have selected a number of potential candidate markers as a result of the extensive literature search. Finally, we have constructed a panel of 224 candidate CE European-specific AIMs. The panel was analysed using Ion AmpliSeq technology and Ion S5 sequencing system. We have sequenced DNA samples from 1600 individuals from Slavic populations of Europe (Poles, Russians, Belarusians, Ukrainians, Czechs, Slovaks and Serbs) as well as populations of different ancestry (Austrians, Arabs, Italians and Tatars). The final step was the construction of an online tool utilizing three different population assignment methods (LR, STRUCTURE and GPS). This tool utilizes the selected SNP set to ascribe unknown samples to a given population or geographical region, with emphasis on potential CE European origin of a sample.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 23 KEYNOTE LECTURE

O013 - EXPLAINING BAYESIAN INFERENCE PRINCIPLES NONVERBALLY: HOW TO HELP NON-MATHEMATICIANS UNDERSTANDING THE WEIGHT OF EVIDENCE Halina Šimková1

1 Charles University, Institute of Criminalistics, Prague, Czech Republic

Thanks to its great potential and sheer versatility, Bayesian inference has become the most progressive tool of the modern era of data science and it is used to deal with various problems in more and more areas of practical life. However, Bayesian inference should not be seen as a mere statistical tool, but rather as one of the fundamental components of general logic and, as such, it should be properly understood by experts in many different fields. Collaboration between mathematicians and non-mathematicians is therefore crucial if the potential of this instrument is to be fully exploited, but this collaboration is often limited by the mathematical anxiety of non-mathematicians. We find ourselves in a situation where many of the people on whom it depends whether the whole process is successful (i.e. lawyers, doctors) are not able to adopt Bayesian inference through classical theoretical mathematics. Fortunately, there are very effective non-verbal ways to explain Bayesian inference that work with visual models built on well-known and intuitively easy-to-understand objects. The lecture presents two of them. First, a WATERFALL MODEL that works with a basic form of Bayes rule and explains the principles of classical test parameters, such as sensitivity, specificity and prior probability, while illustrating the impact of changing these parameters on the test predictive values and the weight of evidence. Second, a SCALES-OF-JUSTICE MODEL, that illustrates an odds form of Bayes rule and it allows for an intuitive understanding of the effect of combining priors and weight of evidence.

24 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O014 - HOW TO AVOID DRIVING DNA CASEWORKERS CRAZY: CASESOLVER, AN EXPERT SYSTEM TO INVESTIGATE COMPLEX CRIME SCENES Lourdes Prieto1, Peter Gill2,3, Øyvind Bleka2

1 Forensic Sciences Institute. University of Santiago de Compostela, Forensic Genetics, Santiago de Compostela, Spain 2 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway 3 University of Oslo, Institute of Clinical Medicine, Oslo, Norway

DNA analyses can be used for both investigative (crime-focused), or evaluative (suspect-focused) reporting. Investigative, DNA-led exploration of serious crimes always involves the comparison of hundreds of biological samples submitted by the authorities for analysis. Crime stain comparisons include both evidence to evidence profiles and reference to evidence profiles. When many complex DNA results (mixtures, LTDNA samples) are involved in the investigation of a crime, the manual comparison of DNA profiles is very time-consuming and prone to manual errors. In addition, if the person of interest is a minor contributor, the classical approach of performing searches in national DNA databases is problematic because it is realistically restricted to clear major contributors, and the occurrence of masking and drop-out means that there will not a definitive DNA profile to perform the search. CaseSolver is an open source expert system that automates analysis of complex cases. It does this by three sequential steps: a) simple allele comparison b) likelihood ratio based on a qualitative model (Forensim) c) likelihood ratio based on a quantitative model (EuroForMix). The software generates a list of potential match candidates, ranked according to the likelihood ratios, which can be exported as a report. In addition, an informative graphical network plot is generated that easily identifies contributors in common to multiple stains. Here we provide a demonstration of a challenging case with the analysis of 119 different crime-stains resulting in mixtures of varying complexity. We also show how we can identify contributors from a database of 1 mill individuals which took about 8.5 hours for our example.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 25 O015 - COMPARISON OF CE AND MPS BASED ANALYSIS FOR THE PROBABILISTIC INTERPRETATION OF MIXED STR PROFILES Corina Benschop1, Kristiaan van der Gaag1, Jennifer de Vreede1, Alwart Nijveld1, Anouk Backx1, Rick de Leeuw2, Sofia Zuniga1, Peter de Knijff2, Titia Sijen1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands 2 Leiden University Medical Center, Department of Human Genetics, Leiden, Netherlands

The interpretation of short tandem repeat (STR) profiles can be challenging when, for example, alleles are masked due to allele sharing among contributors and/or when they are subject to drop-out, for instance from sample degradation. Mixture interpretation can be improved by increasing the number of STRs and/or loci with a higher discriminatory power. Both capillary electrophoresis (CE, 6-dye) and massively parallel sequencing (MPS) provide a platform for analysing relatively large numbers of autosomal STRs. In addition, MPS enables distinguishing between sequence variants, resulting in enlarged discriminatory power. Also, MPS allows for small amplicon sizes for all loci as spacing is not an issue, which is beneficial with degraded DNA. In this study, the performance of the PowerPlex® Fusion 6C STR kit, the MPS PowerSeq™ Auto System and the ForenSeq™ DNA Signature Prep Kit were compared. To that aim, purposeful two- to five-person mixtures varying for mixture proportion, level of drop-out and allele sharing were amplified in triplicate. Profiling results were compared regarding the number of unique alleles, percentage of detected alleles and drop-in. The two-, three- and four-person mixtures were used for likelihood ratio calculations using LRmix Studio and EuroForMix. Analyses were performed using true and non-contributors and results show amongst others the Type I and Type II errors, the effect of drop-out and the effect of replicates. This study shows the types of samples and hypotheses for which either MPS or the use of peak height information yields a stronger weight of evidence. Reversely, we also note those cases in which they do not outperform CE or a semi-continuous model.

26 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O016 - USING GENETIC COMPLEXITY TO SOLVE FORENSIC COMPLEXITY: A NEW CLASS OF COMPLEX HYPERVARIABLE STR MARKERS FOR DECONVOLUTION OF COMPLEX DNA MIXTURES Arwin Ralf1, Leroy de Visser1, Diego Montiel González1, Manfred Kayser1

1 Erasmus MC University Medical Center Rotterdam, Genetic Identification, Rotterdam, Netherlands

DNA mixtures pose a major challenge to routine forensic DNA analysis for individual identification purposes. Despite new software solutions and new biomarkers (such as DIP-STRs) being introduced, many forensic DNA mixtures remain impossible to deconvolute because of the limitations of these approaches. MPS-based analysis of standard forensic STRs improves mixture deconvolution somewhat, because sequence read quantification is more accurate than semi-quantitative CE-based fragment length analysis and MPS allows the detection of micro- variation, but has limitations too. Mixture deconvolution problems using available methods are increased the more persons contributed to a forensic stain (complex mixtures). One of the limitations comes from the degree of polymorphism of the STR markers used, which is sufficiently high for individual identification from single source samples, but not high enough for mixture deconvolution. Aiming at establishing a solution to forensic DNA mixture deconvolution, we identified various extremely variable, highly-complex autosomal STRs that are characterized by multiple long and interrupted repeat arrays. Due to limitations of all forensically used MPS platforms to sequence long DNA fragments, we used the Pacific Biosciences (PacBio) Sequel system that is particularly suitable for high-quality long read sequencing, but thus far has not been applied in forensic genetics. In this proof-of-concept presentation, we introduce this forensically novel class of highly complex, hypervariable STR markers and demonstrate their potential for deconvoluting DNA mixtures, including complex ones, via long read DNA sequencing.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 27 O017 - THE FIRST MPS-STR BASED CONVICTION IN A CRIMINAL CASE? Peter De Knijff1, Rick de Leeuw1, Denise de Wit1, Thirsa Kraaijenbrink1

1 Leiden University Medical Center, Human Genetics, Leiden, Netherlands

On January 17, 2019, Mr. X was convicted by the appeal Court of Amsterdam, The Netherlands, for raping Mrs. Z. Initially, Mr. X was acquitted by the district court of Amsterdam because of inconclusive DNA-evidence obtained by means of capillary electrophoresis of short tandem repeats (CE-STRs). What made this conviction unique in an International perspective, is that this is, as far as we know, the first conviction based on the combined use of massively parallel sequencing of STRs (MPS-STR) and the use of a probabilistic model instead of a consensus approach to interpret the complex (three persons) mixed DNA-profiles. The initial CE-STR analyses were complicated due to (i) the very minor contribution of the suspect’s DNA to all crime-scene samples (between 5 % and 10 %), (ii) masking of 52 % (23 out of 44) of all the STR alleles of the suspect by those of the victim or the victim’s partner, (iii) overlap of 14 % (6 out of 44) of the suspect’s alleles with stutter positions of the alleles of the victim or the victim’s partner, and (iv) absence of several of the alleles that were unique for the suspect in CE-profiles. By means of MPS, using Promega’s PowerSeq 46GY system and the MiSeq FGx from Verogen we could alleviate some of these complications, and obtain STR profiles of a much better quality enabling the detection of a larger number of alleles that were unique for the suspect. The combined effect of this resulted in highly incriminating DNA-evidence in six out of eight different crime-scene samples. We will discuss these MPS-results in comparison with the initial CE-results, and comment on the benefit of using a probabilistic model instead of a consensus approach to interpret complex mixed DNA-profiles.

28 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O018 - ENHANCING STR SEQUENCE ALLELE REPRESENTATION FOR PROBABILISTIC GENOTYPING Rebecca Just1, Jennifer Le1, Jodi Irwin1

1 FBI Laboratory, DNA Support Unit, Quantico, USA

The longest uninterrupted stretch (LUS) concept for STR sequence allele designation was con- ceived to facilitate interpretation of NGS-based STR typing results using current or near-term probabilistic genotyping programs. As the length of the LUS reference region is determined directly from an allele sequence, allele designations are based on the underlying biology of the STR and maintain a clear relationship between parent alleles and stutter products for fully continuous probabilistic genotyping. To increase the number of distinct aSTR alleles that can be represented using the format, we surveyed a published dataset of sequence alleles for 777 individuals from four U.S. population groups. For nine CODIS loci, we identified additional reference regions whose length could be used in allele designations along with the LUS. When combined with the LUS alleles, use of these additional reference regions enabled unique representation of each distinct ForenSeq UAS (Verogen, Inc.) sequence allele observed among the 777 individuals. Population data from seven additional published studies (N=1291 individuals) were subsequently examined to test the performance of the additional reference regions. In these data – originating from 11 population groups across four continents – we identified just eight instances in which aSTR alleles distinct by sequence could not be uniquely represented by use of the LUS combined with the additional reference regions. Extension of the LUS concept to additional reference regions thus captures nearly all UAS sequence variation for the 27 ForenSeq aSTR loci. Allele frequency tables have been generated to assist labs with probabilistic interpretation of NGS data in EuroForMix and other programs that can accommodate the allele designation scheme.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 29 O019 - A MASSIVELY PARALLEL SEQUENCING ASSAY OF MICROHAPLOTYPES FOR MIXTURE DECONVOLUTION Fabio Oldoni1, Drew Bader1, Chiara Fantinato1, Sharon Wootton2, Robert Lagacé2, Ryo Hasegawa2, Joseph Chang2, Kenneth Kidd3, Daniele Podini1

1 The George Washington University, Forensic Science, Washington, USA 2 Thermo Fisher Scientific, Thermo Fisher Scientific, San Francisco, USA 3 Yale University, School of Medicine - Genetics, New Haven, USA

Microhaplotypes (MHs) are biomarkers defined by a set of SNPs within < 300 bp displaying multiple allelic combinations1. The absence of stutter, same-sized alleles, low mutation rate make them promising loci for improving human identification, mixture deconvolution2 and ancestry prediction3. Unlike Sanger sequencing, massively parallel sequencing (MPS) allows determining the parental SNP haplotypes by clonal sequencing of each DNA strand. This study evaluated the mixture performance of a 74 MH locus-panel4, 5 on the Ion Torrent S5TM (Thermo Fisher Sci- entific) platform and compared the results to sequence and size-based STR analysis. The panel’s detection limit was tested between 2 ng and 25 pg DNA input. The assay’s performance was evaluated in parallel with GlobalFilerTM kit on capillary electrophoresis (CE) and Precision ID GlobalFilerTM NGS STR Panel v2 on S5TM. To mimic casework-like samples, a series of balanced/imbalanced two-to-five person mixtures was simulated at different contribution ratios and amounts of DNA. The 74-locus assay was sensitive down to 50 pg input DNA. The analysis of CE-based STR mixtures was challenging; however, it was enhanced when STR mixtures were sequenced since additional minor alleles were observed when different from the stutter sequence. For two-person mixtures, full MH minor profile was reported at 1:10 ratio with minimal allele/locus dropout at 20:1, and more significant at higher ratios. For three-to-five-person DNA mixtures, full MH profiles were reported for all minor donors. Finally, MH loci were tested and found amenable to semi-continuous and fully continuous probabilistic genotyping software analysis. These results indicate that MHs can enhance mixture deconvolution by complementing size and sequence-based STR analysis.

30 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O020 - A TOP-DOWN APPROACH TO MIXTURE EVALUATION Klaas Slooten1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

Presently there exist many different models for determining, in the form of a likelihood ratio, whether there is evidence that a person of interest contributed to a mixed trace profile. These methods have in common that they model the whole trace, hence all its contributors, which leads to the computation time being mostly determined by the number of contributors that is assumed. At some point, these calculations are no longer feasible. We present another approach, in which we serially target the contributors to the mixture according to the proportion of their contribution. This means that any trace can be subjected to calculations, until either (i) evidence in favour of being one of the contributors is found, (ii) no evidence of being among the most prominent contributors is found, or (iii) no evidence of being a contributor at all is obtained. the calculation time now depends on how many contributors are queried independently of the trace's complexity. We do so without using a quantitative peak height model, but only using as assumption that contributors with a larger contribution tend to have higher peaks in the DNA profile of the trace. We show that our approach gives results that are, for the most prominent donor, a very close approximation to the results of a continuous model (regardless of the total number of contributors). For the remaining donors, as we query less and less prominent contributors, our method becomes more conservative with respect to the one of a continuous model . We present results on sets of mixtures, analyzing traces with plausibly 6 or more profile contributors.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 31 O021 - FROM REFERENCE TO MIXTURE TO MIXTURE TO MIXTURE AND BEYOND Maarten Kruijver1, Duncan Taylor2, Jo-Anne Bright1, Emily Rowe2, Damien Abarno2, Zane Kerr1, John Buckleton1

1 Institute of Environmental Science and Research, STRmix, Auckland, New Zealand 2 Forensic Science South Australia, STRmix, Adelaide, Australia

Traditionally, a person of interest's reference profile is compared to a DNA mixture to investigate whether or not this person may have contributed to a crime sample. We present a recent development that allows one to compare multiple DNA mixtures against each other to identify potential common contributors and discuss applications that expand the scope of DNA mixture interpretation. Firstly, we explain how the method can be used for intelligence purposes in the absence of a suspect. Specifically, pairwise comparisons of mixtures with unidentified contributors allows linking of crime scenes which can provide valuable intelligence. Secondly, we have tested the method in a quality assurance context investigating the prevalence of sample-to-sample contamination. Finally, we present a novel extension of the method combining evidence across more than two mixtures simultaneously. This can be used, for instance, to combine the evidence from multiple low level swabs to identify a common contributor where it would not have been possible from the separate samples. Alternatively, the knowledge of common contributor(s) between evidence profiles may be used to improve the genotype resolution of the non-common contributor(s).

32 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O022 - EXPLORING DNA INTERPRETATION SOFTWARE USING THE PROVEDIT DATASET Sarah Riman1, Hari Iyer2, Peter Vallone1

1 National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg, USA 2 National Institute of Standards and Technology, Statistical Design, Analysis and Modeling Group, Gaithersburg, USA

This work explores the effects of variability in the overall DNA typing process on the likelihood ratio (LR) generated from probabilistic genotyping (PG) systems. The data used in this study was obtained from the PROVEDIt set which is a comprehensive dataset openly available at lftdi.com [1]. The selected samples for this study were composed of single-source and complex mixtures amplified with GlobalFiler kit (29 cycles) at varying DNA template amounts and separated on 3500 Genetic Analyzer at different injection times. The PROVEDIt mixture profiles were generated using different genotype combinations of varying ratios and number of contributors. Using fully continuous PG systems, we explore the effects of input DNA quantity, number of contributors, and mixture proportion on the behavior of the calculated LRs. Furthermore, the mixture proportion estimated for each sample is determined and compared to the reported values. 1. Alfonse L.E., Garrett A.D., Lun D.S., Duffy K.R., and Grgicak C.M.: A large-scale dataset of single and mixed-source short tandem repeat profiles to inform human identification strategies: PROVEDIt. Forensic Sci. Int. Genet. 2018; 32: pp. 62–70.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 33 O023 - ARE REPORTED LIKELIHOOD RATIOS WELL CALIBRATED? Jan Hannig1, Sarah Riman2, Peter Vallone2, Hari Iyer3

1 University of North Carolina at Chapel Hill, Statistics and OR, Chapel Hill, USA 2 National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg, USA 3 National Institute of Standards and Technology, Statistical Engineering Division, Gaithersburg, USA

In this work we introduce a new statistical methodology for empirically examining the validity of model-based Likelihood Ratio (LR) systems by applying a general statistical inference approach called generalized fiducial inference [1]. LR systems are gaining widespread acceptance in many forensic disciplines, especially in the interpretation of DNA evidence in the form of probabilistic genotyping systems (PGS). These systems output a Bayes factor, commonly referred to as likelihood ratios in forensic science applications. Methods for examining the validity of such systems is a topic of ongoing interest [2], [3]. In addition to summarizing existing approaches and developing our new approach, we illustrate the methods using the PROVEDIt dataset [4] by examining LR values calculated with open source PG software. [1] Hannig, J., Iyer, H., Lai, R.C.S. and Lee, T.C.M. Generalized Fiducial Inference: A Review and New Results, Journal of the American Statistical Association, 2016, Vol. 111 (515). [2] Brummer, N. Proc. Odyssey 2004 Speaker and Language recognition workshop. ISCA, June 2004, pp. 33–40. [3] Ramos, D. and Gonzalez-Rodriguez J. Forensic Sci Int. 2013 Jul 10;230(1–3):156–69. [4] Alfonse L.E., Garrett A.D., Lun D.S., Duffy K.R., and Grgicak C.M. Forensic Sci. Int. Genet. 2018; 32: pp. 62–70

34 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS KEYNOTE LECTURE

O024 - FORENSIC GENETICS AND DTC GENOMICS: FRIEND OR FOE? Yaniv Erlich1

1 MyHeritage, CSO, Or Yehuda, Israel

Consumer genomics databases reached the scale of millions of individuals. Recently, law enforcement investigators have started to exploit some of these databases to find distant familial relatives. I will present several technical strategies in which law enforcement agencies can leverage genetic tools and internet registries to identify major parts of the US population. However, these enhanced capabilities create for the first time a massive genetic surveillance that can be exploited by bad actors. As such, they already affects the consumer genomics community trust and might create risk for intelligence assets of the US down the road. I will present practical suggestions to mitigate these risks and reconcile the two opposite societal values of genetic privacy and fighting criminals.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 35 O025 - DEVELOPING PRIORITIES FOR DISCUSSION AND OVERSIGHT OF THE RAPIDLY EVOLVING FIELD OF GENETIC GENEALOGY Christopher Phillips1, Rafaela Granja2, Mark A. Jobling3, Erin Murphy4, Debbie Kennett5, Peter Schneider6, Denise Syndercombe Court7

1 Institute of Forensic Sciences- University of Santiago de Compostela, Forensic Genetics Unit, Santiago de Compostela, Spain 2 Institute of Social Sciences, University of Minho, Braga, Portugal 3 Alec Jeffreys Forensic Genomics Unit, Department of Genetics and Genome Biology, Leicester University, Leicester, United Kingdom 4 School of Law, New York University, New York, USA 5 Department of Genetics Evolution and Environment, University College London, London, United Kingdom 6 Institute of Legal Medicine, University Hospital Cologne, Cologne, Germany 7 King's Forensics- Faculty of Life Sciences and Medicine, King's College London, London, United Kingdom

Since April 2018, with the announcement of the arrest of the Golden State Killer suspect, the application of genetic genealogy to unsolved criminal investigations and identification of missing persons has evolved rapidly and has seemingly unfolded without the public’s consent for frameworks to control police use of genetic genealogy databases. This has clear consequences for: public perceptions of established forensic genetic tests; acceptance of new small-scale DNA tests to get additional information from contact traces; existing strict ethical oversight of familial searching of forensic DNA databases; protection of the genetic privacy of those sharing their personal data in community databases to seek relatives; and lack of consent when untested individuals can be identified. As these are areas of critical concern for the ISFG community, we created a group to foster discussion of all aspects of genetic genealogy applied to forensics, keep a watching brief on evolving DNA technologies and help bring consensus on ethical constraints. These efforts are hindered by the fact that almost all DNA analysis techniques applied to genetic genealogy are kept away from independent scrutiny by their commercial protection. For the same reason, proper quality control of laboratory methods generating low coverage sequences or hundreds-of-thousands of SNP genotypes lack independent review to ensure a test’s forensic efficiency and control of contamination. We outline our areas of concern, which focus on: ethical oversight to protect individual privacy and maintain confidence in forensic genetics as a whole; evaluation of the sensitivity, efficiency and limitations of SNP analysis with whole-genome-scans or low coverage sequencing; and full engagement with all stakeholders in this field.

36 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O026 - THE EFFECTIVENESS OF FORENSIC GENEALOGY TECHNIQUES IN THE UNITED KINGDOM – AN EXPERIMENTAL ASSESSMENT Jim Thomson1, Tim Clayton2, John Cleary3, Maurice Gleeson4, Debbie Kennett5, Michelle Leonard6, Donna Rutherford4

1 Eurofins Forensic Services, DNA Research and Development, Teddington, United Kingdom 2 Eurofins Forensic Services, Casework Exam and Reporting, Wakefield, United Kingdom 3 Heriot-Watt University, Languages and Intercultural Studies, Edinburgh, United Kingdom 4 ISOGG UK & Ireland Group, Genetic Genealogist, London, United Kingdom 5 University College London, Genetics- Evolution and Environment, London, United Kingdom 6 ISOGG UK & Ireland Group, Genetic Genealogist, Glasgow, United Kingdom

The use of forensic genealogy techniques to identify Joseph James DeAngelo as the prime suspect in the Golden State Killer case in 2018 has opened up a new approach to investigation of cold cases. Since that breakthrough, these methods have been applied to more than 40 cases with more being reported on a weekly basis. To date, all of these reports relate to investigations in the US, where the high uptake of “direct-to-consumer” (DTC) genetic testing by individuals conducting private ancestral research has provided the necessary publicly available data for successful forensic investigations. We have conducted a study to assess the likely effectiveness of forensic genealogy techniques if applied to investigations in the UK. Volunteers provided their own SNP array data, downloaded from a DTC provider of their choice. These data sets were anonymised and uploaded to the GEDmatch Genesis genealogy website, mimicking data sets from unsourced crime samples. A team of experienced genealogists then attempted to identify the donors of the anonymised data sets by identifying relatives on the database and triangulating to determine their shared family lineages which were further investigated using traditional resources (such as birth, marriage, death and census records). The results of the study will be presented and will provide a valuable indicator of the technical viability of this method in the UK. This can then be considered by policy makers, alongside any perceived ethical, legal and regulatory constraints, in the future planning for use of this technology.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 37 O027 - FORENSIC GENEALOGY – PERFORMANCE OF DENSE SNP DATA TO TRACE DISTANT RELATIVES Daniel Kling1, Andreas Tillmar2

1 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway 2 National Board of Forensic Medicine and Forensic Toxicology, Department of Forensic Genetics, Linköping, Sweden

Forensic genealogy is a concept coined already in 2005. However it has gained increased interest due to the explosion of direct-to-consumer (DTC) tests and in particular following the use of public genealogy databases to trace distant relatives of unknown donors of crime scene samples. In this study we evaluate and present interesting results based on simulated data for some distant relationships. In particular we focus on four different methods to estimate degree of relatedness; Likelihood ratio (LR), Length of shared genomic segments (Segment), Kinship coefficient (Kinship) and Pr(IBD=0). The LR approach is well known in forensics whereas the other methods are more common for denser marker sets and used in other areas of genetics. For instance, DTC companies commonly rely on versions of the Segment approach measuring total length of shared genomic segments based on some thresholds. The results indicate that the traditional LR approach as a single source of classification is as good as, and even better than, the standard IBS approaches. In particular the true classification rate is higher for some distant relationship. However, the LR approach is both computer-intensive and sensitive to population frequencies as well as genetic maps (positions of the markers). We further show that when combining different classification approaches, a lower false classification rate is achieved while still maintaining a high true classification rate. It is crucial that forensic practitioners are aware of the progress and possess a fundamental understanding of the behavior and limitations of the statistical approaches if they are to assist in future endeavors related to forensic genealogy.

38 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O028 - AN INTERNATIONAL CONSIDERATION OF A STANDARDS-BASED APPROACH TO FORENSIC GENETIC GENEALOGY Nathan Scudder1,2, James Robertson3, Sally F Kelty4, Simon J Walsh2, Dennis McNevin5

1 PhD Candidate, University of Canberra, Gungahlin, Australia 2 Australian Federal Police, GPO Box 401, Canberra, Australia 3 University of Canberra, National Centre for Forensic Studies, Canberra, Australia 4 University of Canberra, Centre for Applied Psychology, Canberra, Australia 5 University of Technology Sydney, Centre for Forensic Science, Sydney, Australia

Forensic genetic genealogy has moved into limited operational use in the United States, and received international attention following the arrest of the alleged Golden State Killer. The interest in this emerging area has seen the development of online courses to train investigators to pursue forensic genetic genealogy leads [1] and the emergence of service providers marketing directly at law enforcement [2]. Forensic genetic genealogy is an intelligence capability and can draw on existing intelligence doctrine. The power of genetic genealogy requires consideration of relevant standards, national or international. The development of these standards requires close consideration not just of legal and constitutional issues in the United States, but also privacy and public trust arguments in other countries, including the application of the General Data Protection Regulation in Europe. This presentation examines policy options to best regulate forensic genetic genealogy, and whether existing privacy frameworks are sufficient given individual and family interests in genetic information. We draw upon our extensive research in this field over the last three years [3]. [1] DNA Doe Project 2019, Forensic Genealogy Training, viewed 1 Mar 2019, . [2] P. Armentrout, Parabon® Announces Snapshot® Genetic Genealogy Service for Law Enforce- ment: Forensic DNA Samples Screened for Nearly 100 Agencies and Hits Abound, Parabon® NanoLabs, Reston, VA, 2018. [3] Scudder, N, McNevin, D, Kelty, SF, Funk, C, Walsh, SJ & Robertson, J 2019, 'Policy and regulatory implications of the new frontier of forensic genomics: direct-to-consumer genetic data and genealogy records', Current Issues in Criminal Justice, pp. 1–23.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 39 O029 - WHOLE GENOME SEQUENCING OF HUMAN REMAINS TO ENABLE GENEALOGY DNA DATABASE SEARCHES – A CASE REPORT Andreas Tillmar1,2, Peter Sjölund3, Bo Lundqvist4, Therese Klippmark1, Cajsa Älgenäs1, Henrik Green1,5

1 Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden 2 Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden 3 Peter Sjölund AB, Peter Sjölund AB, Härnösand, Sweden 4 Cold case & Review Team, Regional Investigation Unit, Police Region South Sweden, Swedish Police Authority, Malmö, Sweden 5 Department of Medical and Health Sciences, Linköping University, Linköping, Sweden

Recently a number of high profile crime cases (e.g. the “Golden State Killer”) have successfully been solved or given new leads with the use of genome wide DNA data in combination with pairwise matching from individuals present in genealogy DNA databases. Such databases will primary involve distant relatives which in turn require a large amount of genetic information, in the range of several hundred thousand to millions of SNPs, to be analyzed. While it nowadays is fairly straight forward to obtain such data based on high quality and high quantity DNA, it is still a challenge for degraded DNA of low quantity such in the case of forensic samples. Here we present a successful effort in obtaining genome wide genotype data from human remains. The goal was to identify the remains of an unknown male individual that was found murdered in the south of Sweden in 2003. Whole genome sequencing was performed on DNA originating from a bone sample. Three libraries (replicates) were prepared using ThruPLEX DNA-seq Prep Kit (Rubicon Genomics) which were sequenced on a HiSeq X instrument (Illumina). A mean coverage of 30X was obtained when mapping the sequencing reads to the human genome. Fol- lowing further bioinformatic processing and quality checks, genotypes for approximately one million SNPs were established. The resulting SNP profile was then used for the search for relatives in the genealogy DNA database GEDmatch (www.gedmatch.com). A candidate list of relatives was obtained which was further processed using traditional genealogy methods in order to get investigative leads about the identity of the unknown. In summary, in this case report we show how whole genome sequencing can be used on forensic samples to create the SNP profile required for searches in genealogy DNA databases.

40 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O030 - ETHICAL, SOCIAL AND LEGAL ISSUES OF FAMILIAL SEARCHING: NEW AND OLD DEBATES Rafaela Granja1, Helena Machado1

1 University of Minho, Institute of Social Sciences ICS, Sociology, Braga, Portugal

Familial searching makes use of procedures for detecting genetic relatedness to search for criminal suspects through their connection with relatives. For several years its use has been occurring in exceptional circumstances (i.e. serious criminal cases), relying on a DNA method known as STRs and restricted to government forensic databases. However, recent developments have been outlining how long-range familial searches have been increasingly used by policed forces in commercial recreational genealogy databases, using SNPs. Familial searching thereby currently entails a much wider dispersed network of databases and data subjects and has, consequently, become subject of new, unexpected and still unexplored ethical, legal and social debates. Existing literature on the topic of familial searching is sparse, disjointed and unfocused and still misses an in-depth exploration of the new ethical, legal and social issues that nowadays frame the use of genetic genealogy applied to criminal investigations. As such, the aims of this paper are two-fold: first, it presents an extensive review of literature on the topic of familial searching, with a particular focus on original empirical studies. Secondly, it explores the old and new ethical, social and legal debates framing the use of familial searching in criminal investigation. Issues such as genetic privacy, uncontrolled access by the police to citizens’ genetic data and expansion of affected populations are explored.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 41 O031 - THE GENETIC IDENTIFICATION OF DEAD MIGRANTS IN THE MEDITERRANEAN SEA: THE LAMPEDUSA 2013 SHIPWRECK Barbara Bertoglio1,2, Silvano Presciuttini3, Pierangela Grignani2, Paola Di Simone4, Nicolò Polizzi4, Cristina Cattaneo1, Agata Iadicicco5, Paolo Fattorini6, Carlo Previderè2

1 Università di Milano, LABANOF- Dipartimento di Scienze Biomediche per la Salute- Sezione di Medicina Legale, Milan, Italy 2 Università di Pavia, Dipartimento di Sanità Pubblica- Medicina Sperimentale e Forense, Pavia, Italy 3 Università di Pisa, Dipartimento di Ricerca Traslazionale e Nuove Tecnologie in Medicina e Chirurgia, Pisa, Italy 4 Gabinetto Regionale Polizia Scientifica, Laboratorio di Genetica Forense, Palermo, Italy 5 Ministero dell'Interno, Ufficio del Commissario Straordinario per le Persone Scomparse, Rome, Italy 6 Università di Trieste, Dipartimento Clinico di Scienze mediche- chirurgiche e della salute, Trieste, Italy

On October, 3rd 2013 a boat packed with more than 500 migrants coming from the Horn of Africa sank a few kilometres off the shore of Lampedusa, a small island in the Mediterranean Sea, causing the death of hundreds of persons. Biological samples were collected from the corpses and putative relatives of the victims were reached through an international call. Genetic profiles based on 16 autosomal STRs were obtained. The final genetic database included 363 victims and 43 reference persons from 36 families, missing 35 first degree and 6 second degree relatives. A blind search approach was applied to look for parent-child (PC) and full sibling (FS) pairs, both among the victims and between reference persons and victims. Both the Identity-by-State (IBS) and the Identity by-Descent (IBD) statistics were used. LR and Posterior Probability calculations were carried out by the DVI module of the Familias3 software. The blind search analysis detected some familial groups within the victims, two of which were connected to reference persons. The identifications were confirmed by computing pedigree LRs and posterior probabilities, based on information provided by the relatives. Victim-relative pairs who did not meet the posterior probability threshold required for issuing a match report were further analysed by additional autosomal STRs and lineage markers (Y-STRs and mtDNA). Some critical points emerged, especially concerning the lack of available population data from the Sub-Saharan area and the importance to collaborate with other forensic experts in the identification process. In conclusion, 32 victims were identified by kinship analysis; they included 83 % of the first-degree missing relatives (PC or FS) and 50 % of the second-degree missing relatives.

42 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O032 - RE-EVALUATION OF DNA BASED IDENTIFICATION RESULTS OF VICTIMS OF A TERRORIST ATTACK 25 YEARS LATER Daniel Corach1, Carlos Vullo2, Andrea Sala1, Maria Laura Catelli2, Mariela Caputo1, Magdalena Romero2, Santiago Ginart1, Lucía Garrigos3

1 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Génetica. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG - CONICET., Buenos Aires, Argentina 2 DNA Forensic Laboratory, Argentinean Forensic Anthropology team EAAF, Córdoba, Argentina 3 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Génetica. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG., Buenos Aires, Argentina

In July 1994 there was a second attack against a Jewish institution in Buenos Aires. The target was the Argentine Israelite Mutual Association (AMIA) and the bomb toppled its building and killed 85 people. The Supreme Court ordered to SHDG to carry out the identification of the fragmented remains by means of DNA analysis. At that time, 5 VNTR´s and 7 STR´s were used -5 autosomal, 1 of the X and one of the Y chromosome-, sequencing of the mtDNA HVR I-II and MVRs. The techniques used were all manual and the detection of polymorphisms through radioactive probes or primers. The first report was presented on 11–07–94. In addition to the molecular data, identifications were made using conventional forensic criteria: dactyloscopy, radiography, dentistry and personal traits. In July 2016, the AMIA Investigation Unit, in order to clarify the identity of additional victims, asks SHDG and the Argentinean Forensic Anthropology team (EAAF), as a second opinion lab, to do the analyzes in parallel and compare them with 1994 report. Both laboratories used automated platforms (ABI 3500); commercial kits such us Powerplex Fu- sion and mtDNA sequencing. Results reported in 1994 were confirmed by both laboratories. Additionally, it was possible to identify a victim from material that had been analyzed initially but, given its state of degradation, did not allowed to obtain a genetic profile. The use of high sensitivity megaplexes made it possible to identify a missing person 22 years later. The overall results demonstrate the DNA analysis robustness and the great sensitivity increase of the new analytical platforms and reagents. It also underscored the importance of the inclusion of a second opinion lab to warrant the quality of the results.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 43 O033 - NEW ISO STANDARDS FOR FORENSICS: DNA “FREE” CONSUMABLES AND THE FORENSIC PROCESS Ingo Bastisch1, Linzi Wilson-Wilde2

1 Bundeskriminalamt, KT31, Wiesbaden, Germany 2 National Institute of Forensic Science, Australia New Zealand Policing Advisory Agency, Melbourne, Australia

Ten years ago the case of the German Phantom – a huge police operation that was misled by manufacturing-based contamination of cotton swabs used for trace collection – led to an increased awareness regarding contamination avoidance and detection. One consequence was the development of the ISO standard 18385 that deals with manufacturing requirements to minimize DNA contamination in products used in the forensic DNA analysis process. As a result there are now a variety of products available labelled as “Forensic DNA free” providing increased confidence for forensic users in their testing results. In addition, a further series of ISO standards (ISO 21043) is under development, dealing with requirements for the different steps of the forensic process, starting from recognition and collection at the scene of crime to reporting the results. This set of standards is applicable to all persons and organizations that are part of the forensic process and does not conflict with current accreditation standards like ISO/IEC 17025 or 17020. It is anticipated that these standards will contribute to the international harmonization of processes facilitating acceptance within and between countries. The main aspects of the existing standards will be summarized and the implications for forensic geneticists will be highlighted. In addition, the further standards planned for development will be briefly discussed.

44 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O034 - REFLECTIONS AND EXAMPLES OF PROBLEMATIC REPORTING IN DNA CASES: THE NEED FOR ACCREDITED FORMATS AND CERTIFIED REPORTING COMPETENCE Tacha Hicks1, Alex Biedermann2, Franco Taroni2, Christophe Champod2

1 School of Criminal Justice and Formation continue UNIL EPFL, University of Lausanne, Dorigny, Switzerland 2 School of Criminal Justice, University of Lausanne, Dorigny, Switzerland

Quality assurance is a pre-requisite for all forensic genetic laboratories and professional organisations such as ENFSI, SWGDAM or ISFG. These organisations provide guidelines/ recommendations for the methods used (both for analysis and evaluation), and for reporting. However, there are still many laboratories where evaluation is not yet part of the accreditation. Moreover, proficiency testing on how to report results is still in its infancy. This is problematic as the contribution of forensic science to the justice system depends on how results are conveyed. Here, we discuss examples of examples of written and oral statements and illustrate how accreditation could improve and ensure that guidelines on interpretation are implemented. We underline the need for certifying and validating the knowledge of the experts in that specific field. Indeed, analytical techniques have for many years been the focus of accreditation, rather than the logic of communication of the value of the results obtained with these techniques. It is now time to shift and provide means to ensure that forensic scientists’ conclusions are as robust as the methods.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 45 KEYNOTE LECTURE

O035 - DNA TRANSFER: ASPECTS RELEVANT TO FORENSIC INVESTIGATIONS Roland van Oorschot1

1 Victoria Police, Forensic Services Department, Macleod, Australia

It is becoming increasingly relevant to have a good understanding of DNA transfer, persistence, prevalence and recovery (DNA-TPPR) to assist those requested to provide expert opinion on DNA-related activity-level issues. Understanding the factors influencing DNA-TPPR will also assist in the targeting of samples for collection to provide profiles that will aid forensic investigations, as well as in the application of appropriate contamination minimisation practices that will help protect the integrity of the collected samples and generated profiles. Several factors can impact DNA-TPPR. Some of these will be explored here. Whilst much has been learnt about DNA-TPPR over the past decade or two, a greater investment is required to improve our understanding of some of the factors influencing DNA-TPPR and to generate DNA-TPPR related data that can be readily accessed and utilised by forensic experts. Improvements in additional aspects, such as: the use of appropriate terminology, recognition of the expertise required to perform activity level assessments, and the training and proficiency testing of those assisting forensic investigations and legal deliberations in relation to the transfer of DNA, will aid the ability of forensic experts to provide good quality opinion and guidance to the triers of fact.

46 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O036 - TRANSFER, PERSISTENCE AND RECOVERY OF EPITHELIAL CELLS ON THE SKIN IN DIRECT AND SECONDARY TRANSFER SCENARIOS Ane Elida Fonneløp1, Helen Johannessen1, Arne Roseth1, Ingebjørg Heitmann1, Peter Gill1

1 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway

In court the forensic scientists are often requested to evaluate different propositions with regard to activity level. In order to evaluate the strength of the evidence it is first necessary for the scientist to consider all of the background information in order to formulate his or her expectations of the findings. The victim may provide details of an attack e.g. grabbing of the neck, kissing or biting the victim’s skin. All of these areas can be swabbed in order to search for epithelial cells. Before the results are known, we can surmise that cells from the suspect will either be present or they will be absent with high and low probabilities respectively (the strength of the evidence is measured at sub-source level). Under the defence proposition, if the suspect and victim had previous legitimate contact (e.g. they shook hands or hugged each other) the probability of finding DNA on the swabbed areas (via the mechanism of secondary transfer) will be greater compared to the scenario where they never had contact. In order to evaluate the strength of the evidence at activity level, it is necessary to have knowledge of the probability of DNA recovery given secondary and primary transfer under conditions that simulate the prosecution/ defence propositions. This project comprises experimental data on transfer and persistence to skin, were probabilities are modelled by logistic regression analysis. A Bayesian network is used as the basis to evaluate the strength of evidence that is independent of the findings, and we will provide a Bayesian network for a fictive assault case example.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 47 O037 - MODELLING DNA TRANSFERS IN COMPLEX SCENARIOS Duncan Taylor1, Tacha Hicks2, Christophe Champod2

1 Forensic Science SA, Biology, Adealide, Australia 2 University of Lausanne, Faculty of Law- Criminal Justice and Public Administration, Lausanne, Switzerland

Trace DNA and the manner in which it is transferred from item to item is a common topic arising in forensic science, both in case evaluations, and in Court testimony. In order to assign the probability of obtaining DNA findings, given competing propositions that specify transfer mechanisms, consideration must be given to a number of factors. Previous work has developed a simple Object Oriented Bayesian Network (OOBN) that pooled numerous published studies in order attempt evaluation of trace DNA results given such propositions. In this work, we expand on the previously published OOBN and formalise a number of class networks that can be used together in a logical way to consider DNA movement through complex chains of transfer events. Specifically, we develop an OOBN that considers the two-way transfer of DNA that occurs when two items contact, and allows for the sampling of intermediary items involved in the chain of transfers. We conclude with a demonstration of applying the OOBN being applied to a chain of transfers in a case scenario.

48 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O038 - ASSIGNING FORENSIC BODY FLUIDS TO DNA DONORS IN MIXED SAMPLES BY TARGETED RNA/DNA DEEP SEQUENCING OF CODING REGION SNPS USING ION TORRENT TECHNOLOGY Erin Hanson1, Sabrina Ingold2, Guro Dorum2, Cordula Haas2, Robert Lagacé3, John Ballantyne1,4

1 University of Central Florida, National Center for Forensic Science, Orlando, USA 2 University of Zurich, Zurich Institute of Forensic Medicine, Zurich, Switzerland 3 Thermo Fisher Scientific, Human Identification Research and Development, San Francisco, USA 4 University of Central Florida, Department of Chemistry, Orlando, USA

Biological traces found at crime scenes can be analyzed to genetically identify the donor(s) but also to determine the body fluid composition of the stain. The latter can be accomplished with high specificity by mRNA profiling. In some mixed body fluid samples, it might be of probative value to associate each body fluid with each of the DNA donors. Associating a DNA profile to the contributing body fluid is not often possible, with the exception of simple binary mixtures that contain male and female specific body fluids. However genomic information is transferred from DNA to mRNA, and RNA-cSNPs (coding region SNPs) should be identifiable by MPS methods. Previously, we have performed proof-of-concept studies using a prototype Illumina MiSeq MPS cSNP assay to demonstrate the usefulness of this approach. Here, we have continued our work and introduce an MPS assay on the Ion S5 system comprising a set of 31 cSNPs in body fluid specific mRNA transcripts (7 blood (3 genes); 8 semen (4 genes); 6 saliva (4 genes); 2 vaginal (1 gene); 2 menstrual (1 gene); 6 skin (3 genes)). Combined with optimized primer sets for body fluid identification, the assay can identify all forensically relevant body fluids and skin as well as differentiating blood, semen and saliva transcripts from different individuals. The assay has been evaluated with numerous donors of each body fluid to evaluate the specificity and the discriminatory power of the cSNPs. The findings are promising as we were able to associate donors with body fluids in mixtures of body fluids. However more cSNPs are needed to improve the discriminatory power, particularly for vaginal secretions and menstrual blood. Assigning body fluids to DNA donors is a realistic possibility with the continued development of MPS cSNP assays.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 49 O039 - VISUALISING DNA TRANSFER: LATENT DNA DETECTION USING DIAMOND DYE Jessica Champion1, Roland van Oorschot2, Adrian Linacre1

1 Flinders University, College of Science Engineering, Bedford Park, Australia 2 La Trobe University, Biochemistry and School of Molecular Sciences, Melbourne, Australia

A central question is ‘how did DNA get there’? To help answer this, we visually monitor and record DNA transferring from one substrate to another. When an individual touches a substrate traces of their DNA are transferred (primary) which can then subsequently be transferred to a second substrate (secondary). Currently, DNA transfer and how much remains can only be determined by collecting the biological material from the substrate, isolating the DNA and quan- tifying the amount recovered. However, Diamond™ Dye (DD) enables such DNA transfer events to be visualised by monitoring the movement of cellular material. We examined primary and secondary DNA transfer using four substrates (plastic, aluminium, cotton and polyester) and four contact types between two substrates (passive, pressure, friction and pressure with friction). Participants pressed their index finger against one of the four substrates for 15 s and then DD was applied to the area of contact; cellular material was detected via a fluorescence microscope. Contact between that substrate and a second substrate was performed, using one of the four contact types. After this contact between substrates each substrate was viewed microscopically and transfer of cellular material was recorded. Cellular material could be recorded as having transferred from one substrate to another. Sub- strate and contact type had an effect on the extent DNA transfers. DNA transferred at a higher rate when the first substrate was non-porous than if the first substrate was porous and when pressure with friction was applied than any other contact type. This information expands our understanding of how DNA transfers and which factors affect it, thus assisting greatly with activity level reporting as to how DNA came to be where it was found.

50 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O040 - IN AND OUT OF TOUCH: RELATIVE ACCUMULATION OF CELLULAR AND ACELLULAR “TOUCH DNA” FROM ENDOGENOUS AND EXOGENOUS SOURCES ON HANDS OVER TIME Julie Burrill1, Barbara Daniel1, Nunzianda Frascione1

1 King's College London, King's Forensics, Department of Analytical, Environmental and Forensic Sciences, London, United Kingdom

Little is known about the origins and condition of DNA deposited by handling, also known as “touch DNA,” despite it being regularly analysed in forensic casework. Previous work suggests touch deposits contain both cellular and acellular sources of DNA. The former consists of predominantly anucleate corneocytes previously assumed to have little to no DNA and occasional nucleated cells, while the latter consists of DNA fragments, difficult to amplify. This study examined the relative contributions of each of these fractions to both the hand-endogenous and -exogenous populations by recovering rinses from the inside and outside of worn gloves. Ad- ditionally, cellular and cell-free DNA was collected and measured at time-points up to 2 hours after hand washing without interim contact to assess the relative rates at which each of these fractions may reaccumulate. Microscopic examination was used to visually confirm cell morphology and identify nucleated cell populations. New methods in development for corneocyte lysis and fragmented DNA recovery from touch deposits are also presented here as they were used on cellular and acellular fractions respectively, followed by standard STR profiling. This work introduces the potential value of alternative lysis and purification methods for this sample type in future research. Parsing the contributions to touch deposits and characterizing their DNA content is essential foundational research to fully understand the composition of these increasingly routine samples and how DNA may transfer between items and individuals.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 51 O041 - CHARACTERIZATION OF TISSUE-SPECIFIC BIOMARKERS WITH THE EXPRESSION OF CICRRNAS IN FORENSICALLY RELEVANT BODY FLUIDS Baonian Liu1, Qinrui Yang1, Yuxiang Zhou1, Chengchen Shao1, Yiwen Shen1, Ziqin Zhao1, Qiqun Tang2, jianhui Xie1

1 Fudan University, Department of Forensic Medicine, School of Basic Medical Sciences, Shanghai, China 2 Fudan University, Department of Biochemistry and Molecular Biology, Shanghai, China

Messenger RNA (mRNA) markers have been extensively investigated for the identification of forensically relevant body fluids and tissues based on their expression profiles among cell types. As products of the backsplicing of pre-mRNAs, circular RNAs (circRNAs) share exonic sequences with their linear counterparts. The inclusion of circRNAs in mRNA profiling is shown to facilitate the detection of biomarkers in the identification of body fluids. We identified the expression of circRNAs of 14 out of 45 biomarkers from five body fluid types using outward-facing primer sets and revealed the ratio of circular to total transcripts of biomarkers by RNase R treatment. Furthermore, our results of qPCR analysis show that the inclusion of circRNAs in the detection of biomarkers, including HBA and ALAS2 for blood, MMP7 and MMP10 for menstrual blood, HTN3 for saliva, SPINK5, SERPINB3, ESR1, and CYP2B7P1 for vaginal secretions, TGM4, KLK3, and PRM2 for semen, as well as SLC22A6 and MIOX for urine, does not impair the specificity of these biomarkers. Additionally, a high copy number of targets from linear transcripts could be employed to increase the detection sensitivity of TGM4 and KLK3 with a low expression level of circRNAs in urine samples. Altogether, these results will help with the development of robust multiplex assays for body fluid identification.

52 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O042 - BODY FLUID IDENTIFICATION USING mRNA – BETTER, FASTER, CHEAPER – WHAT METHOD IS BEST OR SHOULD A COMBINATION OF TECHNIQUES BE USED? Rachel Fleming1, Patricia Albani2, Olivia Chirnside2, Anna Lemalu3, Courtney Lynch2, Loren McCarthy2, Jayshree Patel3, Andrew Sarman1, Sallyann Harbison3

1 ESR, Forensic Research and Development, Auckland, New Zealand 2 University of Auckland, School of Chemical Sciences, Auckland, New Zealand 3 ESR, Forensic Biology, Auckland, New Zealand

The source of biological stains in the forensic context is useful to provide information on the nature of a crime, and can be identified using mRNA expression profiling. Since mRNA can degrade over time and due to environmental exposure, it is necessary to use sensitive and stable markers for accurate detection. At ESR, we have been using mRNA to identify body fluids in casework for a number of years using reverse-transcriptase PCR and capillary electrophoresis. We have also been using massively parallel sequencing and other PCR and sequencing techniques to improve, refine and investigate as alternatives to our current mRNA body fluid identification capabilities. This presentation will discuss the biological variation of gene expression, mixed samples and low input samples that are likely to occur in casework with respect to mRNA and what methods work best for different samples. We will discuss the faster, better, cheaper scenario in terms of what this means for implementation of a single technique or combination of methods in operational casework. The difficulties of implementing research into casework using new or a combination of methods will be discussed as well as what is required by both researchers and casework staff to further the implementation of new methods or techniques for body fluid identification into casework.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 53 O043 - PREDICTING THE ORIGIN OF FORENSICALLY RELEVANT BIOLOGICAL MATERIAL USING A MACHINE LEARNING APPROACH Diana Iacob1,2, Angelika Fuerst2, Thorsten Hadrys2

1 Technical University of Munich, Department of Bioinformatics, Munich, Germany 2 Bavarian State Criminal Police Office, Institute of Forensic Sciences, DNA Department, Munich, Germany

DNA profiling alone does not reveal the circumstances by which biological material was deposited and is therefore often not sufficient to accurately determine the nature of a crime. In such cases, the identification of the cellular origin and composition of crime scene related traces, termed body fluid and tissue identification (BFI), can provide contextual information with regards to the circumstances in which the crime unfolded. Our approach uses a targeted mR- NA-Sequencing protocol for body fluid and tissue identification, based on a multiplexed panel of highly specific biomarkers corresponding to the six categories of forensically relevant biological evidences: blood, semen, saliva, vaginal secretions, menstrual blood and skin. Since targeted mRNA-sequencing offers both quantitative and qualitative information it is a very powerful method for RNA profiling. The raw sequencing data was used to build a gene expression pipeline for detecting the expression levels of the biomarkers and their correlations with the body fluids and tissues. Subsequently, the resulting data was used to build a machine learning classi- fier that predicts the origin of single source and mixed samples, respectively. The novelty of this approach consists in incorporating probabilistic information in a machine learning prediction model, rather than having a discrete output.

54 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O044 - DEVELOPMENT OF A MIRNA BODY FLUID PREDICTION SYSTEM USING PROBABILISTIC APPROACHES Zhilong Li1, Xiao Xiao1, Duo Peng1, Huan Tian1, Qian Wang1, Yu Tan1, Weibo Liang1, Lin Zhang1, Peng Bai1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China

Previously, we proposed three miRNAs to distinguish between menstrual blood and peripheral blood samples. In this research, body fluid -specific miRNA markers were screened from three forensically relevant miRNA body fluid datasets (one miRNA sequencing dataset and two miRNA array datasets) to identify five body fluids, namely blood, saliva, semen, vaginal swab and menstrual blood. Two reference RNAs (U6 snRNA and 5s-rRNA) were chosen as single reference gene according to the results of Fujimoto et al. to evaluate the impact of different reference RNAs on miRNA body fluid identification. Body fluid prediction models were trained using Naive Bayes (NB) and Partial least squares discriminant analysis (PLS-DA) respectively. Six blind samples (five single source samples and one mixture sample) were set to test the prediction models. Twelve miRNA markers were confirmed after RT-qPCR detection in 50 samples. Results showed different reference RNAs influenced miRNA expression differences in five body fluids. When 5s-rRNA was used as the reference marker, leave-one-out error of prediction model trained by NB was 0.02 and four components explained 100 % variation of the data. All the five single source blind samples were predicted to be the real body fluid types using NB or PLS-DA. For mixture sample, the prediction model classified it as one of the component body fluids and the posterior probability reduced. To our knowledge, this research was the first attempt to analyze ΔCq value data using probabilistic approaches. The system we proposed here showed probabilistic approach was an improvement of miRNA data interpretation. Prediction models made full use of data to predict the origin of body fluid samples. Further research should focus on mixture studies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 55 O045 - PROTEOMIC GENOTYPING: USING MASS SPECTROMETRY TO INFER SNP GENOTYPES IN A FORENSIC CONTEXT Glendon Parker1, Zachary Goecker1, Jennifer Milan2, Christina De Leon2, Ashleigh Matzoll2, Trevor Borja2, Robert Rice1

1 University of California- Davis, Environmental Toxicology, Davis, USA 2 University of California- Davis, Forensic Science Graduate Program, Davis, USA

Proteins are an intrinsic part of biological evidence. In the last two decades mass spectrometry has revolutionized the analysis of proteins, allowing for thousands of peptides to be detected in a single analysis, including peptides containing single amino acid polymorphisms. Identification of these genetically variant peptides (GVP) allows for inference of the underlying non-synonymous SNP genotype. We have focused on hair proteins as a source of genetic information, with 240 genetically GVPs being discovered and validated. Using optimized processing a single hair shaft can obtain random match probabilities (RMPs) of up to 1 in 100 million. We show that GVP-inferred genotypes are not affected by the anatomical origin of the hair shaft or pigmentation. Harsh oxidation with peroxide does not affect RMPs. Importantly, GVP-inferred SNP genotypes are statistically compatible with STR-typing and Aluretroelements, with 90 % of GVP-inferred SNPs being located greater than 10 million and 75 million bp from the nearest STR or Aluelement respectively. We have shown separation of DNA and peptide workflows from the same samples. Recent work has shifted to the use of a targeted peptide assay. When data acquisition of GVPs was dynamically triggered by the appearance of a standard peptide this approach resulted in a 2.5 fold increase in sensitivity and increased confidence in peptide detection. Based on Markov Chain Monte Carlo modeling we predict this increase in sensitivity will result in RMPs in excess of 1 in 1 trillion from a single hair shaft. Proteomic genotyping can also be applied to any protein matrix, including fingermarks and bone. Proteomic genotyping therefore has potential to complement partial PCR-based DNA typing and provide other options for investigators.

56 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS KEYNOTE LECTURE

O046 - BACKGROUND SELECTION AND BIASED GENE CONVERSION AFFECT MORE THAN 95 % OF THE HUMAN GENOME AND BIAS DEMOGRAPHIC INFERENCES Pouyet, Fanny1

1 Institute of Ecology and Evolution, University of Bern, Bern, Switzerland

Genomic diversity evolves under evolutionary factors such as mutations, selection, gene flow and demography. Across a genome, it varies with recombination in complex ways. For instance, purifying selection is expected to remove diversity with decreasing recombination rate when neutral diversity linked with deleterious variants is removed (background selection, BGS). In this talk we offer to disentangle the effect on genomic diversity of natural selection from that of demography to properly reconstruct the history of species. By using the highquality human genomic data, we show that BGS and a second mechanism determined by base composition, termed GC-biased gene conversion (gBGC) together affect as much as 95 % of the variants of our genome. Interestingly, synonymous sites and nontranscribed regions are also affected, albeit to different degrees such that their use for demographic inference can lead to strong biases. However, this talk illustrates the possibility to identify a set of SNPs that is mostly unaffected by BGS or gBGC, by conditioning on genomic regions with recombination rates above 1.5 cM/Mb and mutation types (C↔G, A↔T). We will show that this neutral set of variants avoids these biases in the reconstruction of human history. If time permits, we will discuss other intricate links between recombination and evolutionary processes across the genome, especially in regions of very low recombination.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 57 O047 - Y-PROFILE EVIDENCE: CLOSE PATERNAL RELATIVES AND MIXTURES Mikkel Meyer Andersen1,2, David Balding3,4

1 Aalborg University, Department of Mathematical Sciences, Aalborg, Denmark 2 University of Copenhagen, Section of Forensic Genetics, Copenhagen, Denmark 3 University of Melbourne, Melbourne Integrative Genomics, Melbourne, Australia 4 University College London, Genetics- Evolution and Environment, London, United Kingdom

We recently introduced a new approach to the evaluation of weight of evidence (WoE) for Y-chromosome profiles by using simulation to describe the distribution of the number of males in the population with a matching Y-profile, both the unconditional distribution and conditional on a database frequency of the profile. Here we extend the approach in two important directions. Firstly, forensic databases are not the only source of background data relevant to the evaluation of Y-profile evidence: in many cases the Y-profiles of one or more relatives of the accused are also available. To date it has been unclear how to use this additional information, but in our simulation-based approach its effect is readily incorporated. We describe this approach and illustrate how the WoE that a man was the source of an observed Y-profile changes when the Y-profiles of some of his male-line relatives are also available. We also demonstrate how these approaches can be applied in casework. Secondly, we extend our new approach to mixtures of Y-profiles from two or more males. Surprisingly, our simulation-based approach reveals that observing a 2-male mixture that includes an alleged contributor's profile is almost as strong evidence as observing a matching single-contributor evidence sample.

58 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O048 - INFERENCE OF ADMIXED ANCESTRY WITH ANCESTRY INFORMATIVE MARKERS Torben Tvedebrink1, Poul Svante Eriksen1

1 Aalborg University, Department of Mathematical Sciences, Aalborg, Denmark

Analysis of Ancestry Informative Markers (AIMs) is of interest to forensic geneticists for the purpose of assessing the origin of individuals. Often AIMs are used to produce investigative leads for potential suspects in crime cases and identification in missing persons mass disaster cases. We have previously suggested to assess the origin of the profile of interest by a likelihood ratio test (LRT) based on Fisher’s exact test. The LRT assesses the likelihood of the data under two competing hypotheses, where the null hypothesis assume common origin of the profile of interest and those in a specified population. However, in the case of a profile with admixed ancestry, there is an increased risk that the profile will be rejected in the constituting populations. The LRT approach is extended by allowing for first- and second-order profile admixture implying the profile of interest may inherit genetic material from up to four populations. The Expec- tation-Maximisation (EM) algorithm is used to handle the ambiguity of allocation of alleles to the proposed populations of origin. To demonstrate the approach, admixed profiles are simulated using AIMs genotypes in eight global meta populations. For certain pedigrees, the ancestral proportions in the offspring are equal for first- and second-order admixtures. Therefore a post- hoc hypothesis test is used to assess if the admixture is of first- or second-order. The methods are implemented in the freely available online software GenoGeographer.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 59 O049 - THE IMPACT OF IGNORING INBREEDING IN KINSHIP EVALUATIONS Hilde Kjelgaard Brustad1, Thore Egeland1

1 Norwegian University of Life Sciences, Faculty of Chemistry- Biotechnology and Food Science, Aas, Norway

Assuming absence of inbreeding within a pedigree is the usual practice of relatedness inference in forensic. This assumption simplifies computations and visualization of relatedness, but conclusions may not be accurate. The IBD (identical-by-descent) parameters describe the relatedness between two non-inbred individuals. More precisely, the probability that for a specific locus, the individuals have zero, one or two pairs of alleles IBD. Based on genetic data from two genotyped individuals, without any prior knowledge of a pedigree connecting them, values of the IBD parameters can be estimated. The IBD parameters are only defined for two non-inbred individuals. If the individuals are inbred, the expected fraction of the IBD region between the individuals increases. Estimation of the IBD parameters then make the individuals seem closer related. For instance by ignoring inbreeding between first cousins, they may appear to be half siblings. As an alternative, the relatedness between two inbred individuals can be described by the Jac- quard coefficients. These nine coefficients give the probability for each possible IBD combination of alleles, at a specific locus for two individuals. For two non-inbred individuals, the first six Jacquard coefficients are zero, and the last three remaining coefficients are equivalent to the IBD parameters. Ignoring the presence of inbreeding also affect the estimated likelihood of the pedigree. By assuming a false non-inbred pedigree connecting two individuals, the likelihood of the pedigree is typically underestimated, compared to the likelihood of the true inbred pedigree. We address the error introduced by assuming no inbreeding between individuals, and how the Jacquard coefficients better can describe the relatedness between two individuals.

60 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O050 - STR SEQUENCE NOMENCLATURE: PROGRESS REPORT FROM THE STRAND WORKING GROUP Katherine Gettings1, David Ballard2, Martin Bodner3, Lisa Borsuk1, Jonathan King4, Walther Parson3, Christopher Phillips5

1 National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg, USA 2 King’s College London, King’s Forensics, London, United Kingdom 3 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 4 University of North Texas Health Science Center, Center for Human Identification, Fort Worth, USA 5 Institute of Forensic Sciences USC, Forensic Genetics Unit, Santiago de Compostela, Spain

Since the 2016 publication of minimal nomenclature requirements from the DNA commission of the ISFG [1], the ad hoc STR Sequence Working Group has been collaborating to harmonize related efforts across our respective laboratories: STRidER STR sequence quality control [2], STRSeq catalog of sequences [3], STRait Razor bioinformatic freeware [4], and the Forensic STR Sequence Guide [5]. A frequent topic discussed among the collaborators is STR sequence nomenclature, as it relates to each laboratory’s efforts and more broadly: lack of nomenclature is often named as a road- block to implementing STR sequencing, and the commercial sector cannot be expected to develop bioinformatic tools for reporting when formal recommendations have not yet arisen from our community. With a goal of expanding and advancing this discussion, in early 2019 this group of collaborators formalized as the STRAND Working Group and received endorsement from the ISFG Executive Board to organize a meeting focusing on STR sequence nomenclature. This meeting, held in April 2019, provided a forum for individuals developing nomenclature schema to present and discuss their ideas, allowing for mutual awareness, identification of differences in approaches, opposing aspects, and opportunities for parallelization while some approaches are still under development. With this presentation, ISFG Congress attendees will be given summary of content from this STR sequence nomenclature meeting, and a path forward will be presented for discussion. 1. Parson W, et al. FSIG. 2016; 22:54–63. 2. Bodner M, et al. FSIG. 2016; 24:97–102. 3. Gettings KB, et al. FSIG. 2017; 31:111–117. 4. King JL, et al. FSIG. 2017; 29:21–28. 5. Phillips C, et al. FSIG. 2018; 34:162–169.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 61 O051 - THE STRidER REPORT ON QUALITY CONTROL OF AUTOSOMAL STR DATASETS – THE GOOD, THE NOT SO GOOD AND THE UGLY Martin Bodner1, Walther Parson1,2, The dna.bases Consortium

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Forensic Science Program, The Pennsylvania State University, University Park, PA, USA

STRidER, the STRs for Identity ENFSI Reference Database (https://strider.online), is a curated, publicly available online allele frequency database, quality control (QC) and software platform for autosomal STRs developed in agreement with the DNA Commission of the ISFG in 2016. Contin- uous updates comprise additional STR loci and populations in the frequency database, the Fo- rensic STR Sequence Structure Guide and further STR-related aspects. One significant innovation by STRidER is the autosomal STR data QC provided prior to publication of datasets. Such scrutiny was lacking previously, leaving QC to authors, reviewers and editors. Results from QC of the >150 STR datasets containing >150,000 individual genotypes submitted since 2017, when the leading forensic genetics journal, FSI:Genetics, made STRidER QC mandatory, will be discussed. Our experience clearly shows that centralized QC and data curation is essential to maintain the high standard required in forensic genetics. This major benefit that STRidER is providing to the scientific community is highlighted by an unexpectedly high rejection rate and virtual absence of error-free submissions. While many errors had minor impact on the resulting allele frequencies that would ultimately be relied upon in forensic casework, there were multiple errors commonly found in single datasets. In addition, several datasets containing serious flaws were observed. The dna.bases project was supported by HOME/2010/ISEC/MO/4000001759 (STEOFRAE) and is co-funded by the Internal Security Program of the European Union (ISFP-2016-AG-IBA-ENFSI). This work received support from European Union grant agreement no. 779485-STEFA-ISFP-2016-AG-IBA- ENFSI.

62 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O052 - ADVANCING MITOCHONDRIAL GENOME DATA INTERPRETATION IN MISSING PERSONS CASEWORK Charla Marshall1,2,3, Kimberly Sturk-Andreaggi1,2, Joseph Ring1,2, Cassandra Taylor1,2, Suzanne Barritt‑Ross1,2, Walther Parson3,4, Timothy P. McMahon1

1 Armed Forces Medical Examiner System, Armed Forces DNA Identification Laboratory, Dover AFB, Delaware, USA 2 SNA International, Alexandria, Virginia, USA 3 The Pennsylvania State University, Forensic Science Program, State College, Pennsylvania, USA 4 Medical University of Innsbruck, Institute of Legal Medicine, Innsbruck, Austria

In recent years, forensic scientists have implemented massively parallel sequencing of the complete mitochondrial genome for forensic applications, such as missing persons casework. While enhanced discrimination power over the commonly targeted control region is an immediate advantage, the use of the complete mitochondrial genome remains hindered by insufficient population data and exclusion/match criteria. To this end, the Armed Forces Medical Examin- er System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL) has developed a high capacity mitochondrial genome sequencing workflow for reference sample databasing. In a three-year project funded by the National Institute of Justice, the AFMES-AFDIL will generate 4,000 United States and 1,000 global mitochondrial genome sequences to be deposited in EM- POP. Results from the initial 2,000 samples indicate a first-pass success rate of 80 %, with >94 % unique haplotypes. Additionally, in an effort to characterize the expected number of differences in mitochondrial genome sequences between maternal relatives, over 600 CEPH family pedigree samples from 87 lineages were processed using the high throughput method. Although stochastic artifacts in the CEPH cell lines elevated the point heteroplasmy rate three-fold compared to population sample data (1.8 vs. 0.46 point heteroplasmies per individual, on average), the CEPH family sequences exhibited a maximum of two substitutions between any two maternal relatives in over 500 generational events. The family study furthermore showed that elevat- ing the variant detection threshold may create false substitutions at heteroplasmic sites, which could lead to false exclusions. Altogether these data support the interpretation of mitochondrial genome sequences in missing persons casework.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 63 O053 - CSY? A PANEL-BASED MPS APPROACH INCLUDING 12,523 Y-CHROMOSOME POLYMORPHISMS Sofie Claerhout1, Paulien Verstraete1, Maarten H.D. Larmuseau1,2,3, Ronny Decorte1,4

1 KU Leuven, Forensic Biomedical Sciences, Forensic Genetics and Molecular Archaeology, Leuven, Belgium 2 Histories vzw, Heemkunde - genealogie, Mechelen, Belgium 3 KU Leuven, Laboratory of Socioecology and Social Evolution, Department of Biology, Leuven, Belgium 4 University Hospital Leuven, Laboratory of Forensic Genetics and Molecular Archaeology, Department of Forensic Medicine, Leuven, Belgium

Until now, the golden standard to genotype Y-STR alleles in forensic DNA investigation relies on fragment analysis by capillary electrophoresis (CE). Unfortunately, it does not take variance on the DNA sequence into account which could be useful for forensic science. Massive parallel sequencing (MPS) is a high-throughput sequencing technique which can expose previously hidden mutations, enabling to study this on another level. The rapid advancement of MPS technology in the last decade has revolutionized genetic research and provides the opportunity to genotype simultaneous hundreds of polymorphisms of interest in a single assay on multiple samples. This study focusses on the development of a panel-based targeted resequencing approach consisting of phylogenetic informative Y-SNPs and multiple Y-STRs for genotyping in a single assay. Our panel targets 857 fragments (average size of 248 base pairs) containing 12,259 Y-SNPs and 264 Y-STR loci (including 15 Y-STRs from the commercially available kits). Primer pairs were designed with DesignStudio (Illumina) for the TruSeq Custom Amplicon Low Input Kit (Illumina). Libraries were dual index labeled and paired-end sequenced (2x300 bp) on the MiSeq platform (Illumina). Data was analyzed using Yleaf Software for Y-SNPs whereby 1,212 deep Y-SNP subhaplogroups (covering 96 % of the minimal Y tree composed of Van Oven et al.) could be defined and through STRait Razor v3 for Y-STR allelic variation. During this presentation, we will discuss the performance of this novel MPS approach in the analysis of gene- alogically related males.

64 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O054 - ANALYSIS OF RECOMBINATION AND MUTATION EVENTS FOR 12 X-CHR STR LOCI: A COLLABORATIVE FAMILY STUDY OF THE ITALIAN SPEAKING WORKING GROUP GE.F.I. Carla Bini1, Ciro Di Nunzio2, Serena Aneli3, Stefania Sarno4, Milena Alù5, Eugenia Carnevali6, Emma Colao7, Michele Di Nunzio8, Matteo Fabbri9, Paolo Fattorini10, Pierangela Grignani11, Piccinini Andrea12, Elena Ponzano13, Carlo Robino14, Anna Rocchi15, Francesca Scarnicci16, Chiara Turchi17, Andrea Verzeletti18, Susi Pelotti19

1 University of Bologna, DIMEC-Dipartimento di Scienze Mediche e Chirurgiche, Bologna, Italy 2 University Magna Graecia, Department of Legal Medicine, Catanzaro, Italy 3 University of Turin, Department of Medical Sciences, Torino, Italy 4 University of Bologna, BIGEA-Department of Biological, Geological and Environmental Sciences, Bologna, Italy 5 University of Modena and Reggio Emilia, Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy 6 University of Perugia, Department of Biomedical and Surgical Sciences, Section of Legal Medicine and Forensic Science, Perugia, Italy 7 Hospital Agency Materdomini, Department of Medical Genetics, Catanzaro, Italy 8 Biogem Scarl, Forensic Genetics Laboratory, Ariano Irpino AV, Italy 9 University of Ferrara, Department of Public Health, UOL of Legal Medicine, Ferrara, Italy 10 University of Trieste, Department of Medicine, Surgery and Health, Trieste, Italy 11 University of Pavia, Department of Public Health, Experimental and Forensic Medicine, Pavia, Italy 12 University of Milano, Department of Biomedical Sciences for Health Forensic Genetics Laboratory, Milano, Italy 13 University of Padova, Department of Molecular Medicine, Padova, Italy 14 University of Turin, Department of Public Health Sciences and Pediatrics, Torino, Italy 15 University of Pisa, Department of Molecular and Critical Area Surgical Pathology, Pisa, Italy 16 Catholic University of the Sacred Heart, Public Health Institute, Section of Forensic Medicine, Rome, Italy 17 Polytechnic University of Marche, Department of Biomedical Sciences and Public Health, Section of Legal Medicine, Ancona, Italy 18 University of Brescia, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Brescia, Italy 19 University of Bologna, DIMEC- Dipartimento di Scienze Mediche e Chirurgiche, Bologna, Italy

According to ISFG guidelines on the use of X-chromosome, the biostatistical evaluation in kinship analysis is based on a likelihood ratio approach, but since in calculation linkage and recombination events should be accounted for, an accurate estimate of haplotype frequencies of the reference population, as well as the knowledge of mutation and recombination rates of X-markers analyzed are required. The increased demand to forensic laboratories for kinship investigations in complex cases explains the need to enlarge the Italian population database for 12 X-STRs routinely used for forensic applications. Indeed, due to the mode of genetic transmission and physical location, sometimes X-chromosome markers can be more informative than autosomal STRs and their analysis could be considered a supplementary tool in DNA testing. A collaborative exercise involving 17 laboratories of the Italian Speaking Working Group Ge.F.I. was organized to evaluate mutation and recombination events in 12 X-STRs included in the In- vestigator Argus X12. In order to explore the segregation stability, three-generation families (grandpa-mother-son) and two-generation families (mother-sons, father-daughter) were ana-

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 65 lyzed and calculation to estimate the recombination fractions between pairs of markers and mutation rates were performed. Evidence of mutation as well as inter- and intra-cluster recombination events were observed. Moreover, 1042 unrelated male individuals from different regions of Italy were typed to expand the existing Italian X-chromosome haplotype frequencies database. Population genetic parameters of forensic interest and genetic distances between the Italian sample and worldwide populations are reported.

66 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O055 - GENETIC PEOPLING OF PAKISTAN AND THE IMPACT OF HISTORICAL MIGRATIONS, ETHNIC CULTURES AND THE PRACTICE OF ENDOGAMY ON THE FORENSIC MATCH PROBABILITIES Ijaz Anwar1, Franco Taroni1

1 School of Criminal Justice- Batochime- Dorigny 1015-CH, University of Lausanne, Lausanne, Switzerland

The demographic history of Pakistan dates back to 7000 BC. It is the birth place of Harappa, Meh- rgarh and Indus civilizations. It has seen the invasions of Greeks, Arabs, Mongols and the British. Pakistan keeps a unique mélange of local and adopted cultures. Pakistan is also the most con- sanguineous country, where cousin marriages account for more than 50 % of the total unions. In order to study the impact of migrations and endogamy, 1020 individuals belonging to Pun- jabi, Saraiki, Pakhtun and Sindhi populations were analyzed based on 15 autosomal STR loci.

Results showed that mean Observed Heterozygosity (HO) ranged from 0.7267 in Sindhi population to 0.7805 in Pakhtun population, mean Gene Diversity (HS) ranged from 0.7978 in Sindhi to

0.8038 in Saraiki population. Mean values for coefficient of inbreeding (FIS) ranged from 2.29 % to

9.09 % which is quite high for natural human populations. FST based genetic distance was lowest (0.0007) between Pakhtun and Sindhi, and highest (0.0024) between Punjabi and Sindhi populations. In Discriminant Analysis of Principal Components, all four studied populations showed a high degree of admixture. Compoplot analysis demonstrated that it was difficult to assign the ethnic affiliation of an individual to a specific group or population due to genetic similarities among individuals. Phylogenetic tree (UPGMA) and NMDS plot presented all four Pakistani populations in a single cluster. In conclusion, these societies have tried to retain their genetic pool by keeping the tradition of endogamous marriages intact, which is quite evident from high FIS values. This unique behavior advocates the forensic practitioners to use FIS values along with FST when calculating the forensic match probabilities to avoid overstatement of a biological evidence.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 67 O056 - THE SCOPE AND LIMITATIONS OF THE LIKELIHOOD RATIO METHOD APPLIED TO STR DATA FOR DETERMINING GENETIC KINSHIP IN ANCIENT OR ISOLATED HUMAN POPULATIONS Vincent Zvènigorosky1,2, Audrey Sabbagh3, Angéla Gonzalez2, Jean-Luc Fausser2, Eric Crubézy4, Ludes Bertrand1, Keyser Christine1,2

1 Paris Descartes University, De la diversité des populations à l'individu- de l'identification à l'identité CNRS FRE2029, Paris, France 2 University of Strasbourg, Strasbourg Institute of Legal Medicine, Strasbourg, France 3 Université Paris Descartes, Institut de Recherche pour le Développement- Faculté de Pharmacie de Paris, Paris, France 4 University of Toulouse Paul Sabatier, Anthropologie Moléculaire et Imagerie de Synthèse CNRS UMR 5288, Toulouse, France

The Likelihood Ratio (LR) method is commonly used to identify kinship in civil, criminal or forensic cases. During the past fifteen years, our team has also used LR applied to ancient STR data and produced satisfactory results within studies of collections of graves or large necropolises. Although we were able to construct genealogies comprising several individuals, it quickly appeared that some pairs showed ambiguous results. Second-degree relationships, half-sibling pairs for example, were often incoherent with detected first degree relationships, such as parent/child or brother/sister pairs. We therefore implemented a study to provide empirical estimations of the error rates for the LR method in living populations with STR allelic diversities comparable to that of the ancient populations previously studied. We studied 900 pairs of STR profiles from individuals with known relationships from North-East- ern Siberia and West Africa. Because commercial STR panels were constructed for specific regions (namely Europe and the US), their allelic makeup showed a significant deficit in diversity when compared to European populations. We determined the capacity of the LR method to confirm known relationships (efficacy) and its capacity to detect those relationships (reliability). We show that the use of approximated allelic frequencies leads to specific issues (both false positives and false negatives) that prevent the determination of second-degree kinship in small populations. Therefore, in the study of ancient populations or human remains of unknown origins, we recommend a conservative approach to kinship determined by LR. We also discuss the potential of uni-parental markers and Next Generation Sequencing in resolving these problematic cases.

68 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS KEYNOTE LECTURE

O057 - USING MICROBIOME TOOLS TO ESTIMATE THE POSTMORTEM INTERVAL OF HUMAN REMAINS Jessica Metcalf1

1 Department of Animal Sciences, Colorado State University, Fort Collins, Colorado, USA

The co-evolution of microbial decomposers and vertebrate carcasses has resulted in conserved cross-kingdom ecological interactions and metabolic pathways. Therefore, microbial succession during decomposition of vertebrates may be repeatable and generalizable enough to develop predictive tools based on microbiome data. Over the past several years, we have been developing a microbial clock to estimate how long human remains have been decomposing. In a large-scale, collaborative research project, we placed 36 donated human cadavers at three anthropological research facilities, located in distinct geographic regions in the United States (Colorado Mesa University, Sam Houston State University, University of Tennessee Knoxville). At each facility, three bodies were placed outdoors each season for four seasons. Skin and soil swabs were collected daily from each body for 21 days of decomposition. From these swabs, we characterized the decomposer microbial community by amplicon sequencing (16S and 18S rRNA gene) to reveal composition and diversity, shotgun metagenomics to reveal potential gene function, and metabolomics to assess small molecules generated by the microbes. Data were used to train machine learning models to predict the postmortem interval (PMI). 16S rRNA gene amplicon data revealed PMI prediction errors of 2–4 days within each facility over 21 days of decomposition in the spring season, and approximately 3.5 days when all facilities were used in model construction. Models included a temperature-based Accumulated Degree Day (ADD). Additional modeling will include data from summer, fall, and winter seasons, as well as other ‘omic data and variables (e.g. humidity) to generate the most robust predictive model possible for these data. Overall, we demonstrate that microbiome tools provide a potentially powerful new tool for the forensic science community.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 69 O058 - ENVIRONMENTAL DNA TO ASSIST FORENSIC INVESTIGATIONS, COUNTER-TERRORISM, FRAUDULENT MEDICINES AND DRUG SEIZURES Jennifer Young1, Arif Malik1, Sarah Ishak2, Eleanor Dormontt2, Kirstie Scott3, Andrew Lowe2, Adrian Linacre1

1 Flinders University, College of Science and Engineering, Adelaide, Australia 2 University of Adelaide, Advanced DNA- Identification and Forensic Facility- School of Biological Sciences, Adelaide, Australia 3 Liverpool John Moores University, Natural Science and Psychology, Liverpool, United Kingdom

Massive parallel sequencing (MPS) has revolutionised the field of genomics enabling substantial advances in human DNA profiling. On top of this, the advent of MPS allows biological signatures to be obtained from complex DNA mixtures. Many studies have applied MPS technology to characterise the biodiversity within environmental samples (such as soil, water and ice cores) and address questions related to ecology, conservation, climate change and human health. Despite these technological advances, translation of these tools to forensic questions and scenarios, remains in its infancy. Soil DNA profiling has been used for comparative purposes to link a suspect to a scene or victim, and some preliminary studies have explored the use of skin microbiome to identify individuals. Here, we discuss the use of MPS to predict soil provenance on a continental scale for counter-terrorism intelligence purposes. We assess the potential of the plant microbiome to increase species resolution on a local scale and thus individualise trace plant fragments within a single species. We explore the application of diatom DNA analysis to discriminate between close-proximity aquatic environments and compare results to microscopy currently employed. We also explore the detection and recovery of human and non-human DNA from pills to identify different batches, and potentially the source, of fraudulent medicines and drug seizures.

70 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O059 - TAXONOMY-INDEPENDENT DEEP LEARNING MICROBIOME APPROACH FOR ACCURATE CLASSIFICATION OF FORENSICALLY RELEVANT HUMAN BIOMATERIALS USING TARGETED MPS Celia Díez López1, Athina Vidaki1, Arwin Ralf1, Diego Montiel González1, Djawad Radjabzadeh2, Robert Kraaij2,3, André G. Uitterlinden2,3, Cordula Haas4, Oscar Lao5,6, Manfred Kayser1

1 Erasmus University Medical Center, Genetic identification, Rotterdam, Netherlands 2 Erasmus University Medical Center, Internal Medicine, Rotterdam, Netherlands 3 Erasmus University Medical Center, Epidemiology, Rotterdam, Netherlands 4 University of Zurich, Zurich Institute of Forensic Medicine, Zurich, Switzerland 5 Barcelona Institute of Science and Technology BIST, CNAG-CRG- Centre for Genomic Regulation CRG, Barcelona, Spain 6 Universitat Pompeu Fabra UPF, Universitat Pompeu Fabra UPF, Barcelona, Spain

Identifying the body source of human biological traces found at crime scenes is relevant for crime reconstruction; however, the use of human cell biomarkers provides challenges. Em- ploying the human microbiome is expected to be useful, as distinct microbial communities inhabit different body sites. Previous tissue identification attempts with microbial DNA suffered from limited numbers of target species and samples. Here we overcome these limitations by introducing a novel taxonomy-independent deep learning (DL) microbiome approach based on large reference data. The DL was based on two classification algorithms: one for epithelial (skin, saliva, vaginal secretion) and another for blood sources (venous, menstrual, fingerprick). Reference 16S rRNA gene sequencing data of different body sites from the Human Microbiome Project (HMP) and de novo generated MPS data from >1,600 samples were used to train 50 DL networks. The most informative features (inertia >0.6, highly different across body sites) used in the DL were extracted from a Correspondence Analysis. Validation testing in >200 test samples via 16S rRNA gene MPS with the Ion Torrent™ PGM and the Ion S5 System achieved high classification accuracies with AUC values >0.99 for all categories. Additional forensic validation testing in >60 mock casework samples aged 1–8 years revealed similarly accurate results. Compared to previous human mRNA-based test outcomes in the same mock samples, our microbiome DL based approach performed mostly better or similarly good. Our microbiome approach allows accurate body source identification of forensically relevant human biological materials and is suitable for future applications in forensic casework, provided further forensic developmental validation testing being successful.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 71 O060 - PERFORMANCE OF ENVIRONMENTAL DNA METABARCODING IN SOIL TRACE MATCHING AND PROVENANCING Tobias Guldberg Frøslev1, Camilla Fløjgaard2, Ida Broman Nielsen1, Anders Johannes Hansen1

1 University of Copenhagen, Department of Biology- Center for GeoGenetics- Øster Voldgade 5- DK1350, Copenhagen, Denmark 2 Section for Biodiversity, Department of Bioscience- Aarhus University- Grenåvej 14, 8410 Rønde, Denmark

DNA extracted from environmental samples (eDNA) – especially soil – is beginning to find use in forensic case work. As part of the SoilTracker project we evaluated the performance of different molecular marker for matching soil traces on shoes with different potential “crime scenes” in an urban environment, and also for provenancing soil traces of unknown origin. For the matching tests, a clean shoe was worn in each of 6 relatively similar urban habitats, and at each “crime scene” 5 reference samples were taken close to, and samples were investigated by eDNA metabarcoding with 6 different molecular markers (fungi, plants, eukaryotes, nematodes, protozoa and bacteria). All markers could unambiguously be assigned to their matching “crime scenes”. For the provenancing tests we used data from the nation-wide biodiversity study (Biowide), and likewise tested the performance of eDNA metabarcoding of different genetic markers to predict the origin of “unknown” soil samples, and also to predict habitat characteristics (forest, meadow, agriculture, etc..) of the sample origin. Overall, we achieved good predictions of environmental gradients and habitat classes to derive a relatively accurate description of the “crime scene”. Thus, we conclude, that there is a large potential for the use of metabarcoding in matching and provenancing of soil trace samples.

72 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O061 - COLLABORATION ACROSS BORDERS TO IMPLEMENT A EUROPEAN DATABASE FOR CANINE STR IDENTIFICATION MARKERS Federica Giangasparo1, Florence Anderson1, Josephin Heinrich2, David Ballard1, Cordula Berger2, Burkhard Berger2, Brian Catchpole3, Walther Parson2,4, Denise Syndercombe Court1

1 King's College London, King's Forensics, London, United Kingdom 2 Medical University of Innsbruck, Institute of Legal Medicine, Innsbruck, Austria 3 Royal Veterinary College, Pathobiology and Population Sciences, London, United Kingdom 4 The Pennsylvania State University, Forensic Science Program, Pennsylvania, USA

The CaDNAP STR panel has proved useful in the determination of canine identification, with 13 autosomal STR markers and 2 gender markers included within the panel. Moreover, recent interest has been raised regarding the possibility of using the autosomal STR genotypes to infer the breed of origin of the animal. From a collaboration between King’s College London, the Roy- al Veterinary College and the GMI institute of Innsbruck, STR genotype data from UK dogs has been compared with their European counterparts e.g. Austrian, German and Swiss dog populations, with respect to dogs within the same breeding group. Preliminary data has shown good general correlation between STR genotype patterns of dogs within the same breed regardless of country location, suggesting that building a canine European database is possible and advantageous. Some discrepancies have been observed however with specific breeds, indicating that while a European database is certainly possible, and would be of high impact for the community, more data is needed to reliably assess inter-country variation. The need for more samples to be tested and more countries to take part is strong and we would like to encourage the community to invest in our animal friends.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 73 O062 - MULTI-LOCUS DNA METABARCODING FOR AUTHENTICATION OF HIGHLY PROCESSED MEAT PRODUCTS COLLECTED IN SOUTH AFRICA Carlotta Pietroni1,2, Tobias Guldberg Frøslev2, Megan Dawn De Jager1, Julian May3, Leif Petersen3, Anders Johannes Hansen2, Maria Eugenia D’Amato1

1 Forensic DNA Lab, Department of Biotechnology, Cape Town, South Africa 2 Centre for GeoGenetics, Department of Biology, Copenhagen, Denmark 3 DST-NRF Centre of Excellence in Food Security, University of the Western Cape, Cape Town, South Africa

DNA metabarcoding species identification has shown the potential to be applied to uncover the adulteration of food items. The capability of metabarcoding derives from being, in essence, the combination of two technologies: DNA barcoding, that utilises the sequence of specific DNA regions to identify species, and Next-Generation Sequencing. Metabarcoding can be applied to products that contain degraded DNA, such as the samples included in our study, and permits the detection of all species within a sample, providing a tool to describe the multiple ingredients often indistinguishable by morphological analysis in processed food items. Some adjustments of the technique are required when it is applied to a different sample type (e.g. canned items, vegetarian products). In this study, we have tested metabarcoding for authentication of 240 processed meat items collected in South Africa (SA). DNA was isolated by salting out precipitation and amplified with previously published primers targeting the mitochondrial Cytochrome b and c oxidase and 16S ribosomal RNA, the internal transcribed spacer (ITS) 2 and the chloroplast trnL regions. Sequenc- ing was performed using a MiSeq. We aim to investigate the usefulness of applying a metabarcoding workflow on processed meat items, by evaluating the amplification performance of the selected primers and their ability to detect at a species level the sample composition. This study will also provide insight regarding the possible presence of mislabelled meat products in the SA market at the time of the samples’ collection. Further validations as the one proposed are still needed to test metabarcoding applicability and reproducibility with different food types, before that this methodology could be applied as a valid forensic analytical method.

74 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O063 - SPECIES IDENTIFICATION USING MASSIVELY PARALLEL SEQUENCING – DETECTING MULTIPLE SPECIES IN MIXED SOURCES Barbara Dellamico1, Andreas Tillmar1,2

1 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden 2 Linköping University, Department of Clinical and Experimental Medicine, Linköping, Sweden

Species identification can be used to help solving crimes such as poaching of animals and illegal harvesting, as well as to determine if a forensic sample or stain is of human origin or not. Most of the existing DNA typing methods for species identification use species-specific primers and focus on single-species DNA sources. In forensic applications, however, there can be cases were no prior information about the species is available. In addition, there can be cases where all species from mixed sources need to be determined. The aim of this study was to design and develop a universal DNA typing method for species identification, with focus on species belonging to the subphylum Vertebrata, based on massively parallel sequencing (MPS) that has the possibility to detect multiple species in a mixed source. Three mitochondrial DNA targets, 16S ribosomal RNA (16S rRNA), cytochrome b (Cyt b) and the cytochrome c oxidase I (COI), were chosen and universal primers for all three targets were selected based on published studies. In silico analysis of mitochondrial DNA reference sequences from more than 4,500 different species showed that the selected targets will discriminate the vast majority of species. DNA analysis have been performed using the selected universal primers on single source samples, artificial DNA mixtures and samples from authentic forensic casework using the QIAseq 1-Step Amplicon Library Prepa- ration (Qiagen) combined with sequencing on a MiSeq FGx (Illumina). Preliminary results show that species from single DNA sources as well as multiple donors in species-species mixtures can be detected, thus confirming the results from the in silico analysis. Data from sensitivity, robustness and accuracy studies will also be presented and discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 75 O064 - SPECIES IDENTIFICATION IN ROUTINE CASEWORK SAMPLES USING THE SPInDel KIT Filipe Pereira1, Cíntia Alves2, Cátia Couto1, Lourdes López Díaz3, David Parra3, Sandra Furfuro4, Mercedes Aler5, Luis Burillo Borrego6, Tereza Olekšáková7, Filipa Balsa8, Lisa Sampaio8, Maria João Porto8, Heloísa Afonso Costa9, Cristina Arévalo Voss10, Mariela Caputo11, Daniel Corach11, Óscar García12, Susana Pedrosa Moro13, Rui Pereira14, António Amorim14

1 IDENTIFICA, Science and Technology Park of the University of Porto - UPTEC, Porto, Portugal 2 University of Porto, Faculty of Sciences, Porto, Portugal 3 Servicio de Criminalística de la Guardia Civil, Departamento de Medio Ambiente, Madrid, Spain 4 Universidad Nacional de Cuyo Facultad de Ciencias Médicas, Laboratorio de Análisis de ADN, Mendoza, Argentina 5 Servicio de Laboratorio. Instituto de Medicina Legal y Ciencias Forenses, Sección de genetica y Criminalistica, Valencia, Spain 6 Instituto de Medicina Legal y Ciencias Forenses de Valencia, Sección de Biología. Servicio de Laboratorio, Valencia, Spain 7 Institute of Criminalistics, Department of Special Biology, Prague, Czech Republic 8 Delegação do Centro, Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Serviço de Genética e Biologia Forenses, Coimbra, Portugal 9 Delegação do Sul do Instituto Nacional de Medicina Legal e Ciências Forenses, Serviço de Genética e Biologia Forenses, Lisboa, Portugal 10 Comisaría general de Policía Científica- Cuerpo Nacional de Policia Spain, Laboratorio de ADN, Madrid, Spain 11 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología- Inmunología, Biotecnología y Genética -. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG - CONICET., Buenos Aires, Argentina 12 Basque Country Police-Ertzaintza, Forensic Science Unit, Forensic Genetics Section, Erandio- Bizkaia, Spain 13 NASERTIC, Unidad de Laboratorio de Navarra de Servicios y Tecnologías, Villava, Spain 14 Universidade do Porto, Instituto de Investigação e Inovação em Saúde- I3S, Porto, Portugal

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identiﬁcation by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 and 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95 % of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was

76 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 77 O065 - WHOLE-GENOME SEQUENCING OF NEISSERIA GONORRHOEAE IN A FORENSIC TRANSMISSION CASE Carlos Frances‑Cuesta1, Idoia de la Caba2, Idigoras Pedro2, Amparo Fernandez‑Rodriguez3, David del Valle Perez4, Jose Maria Marimon5, Fernando Gonzalez‑Candelas6

1 Universitat de Valencia - FISABIO, Joint Research Unit “Infection and Public Health”, Valencia, Spain 2 Hospital Universitario Donostia, Instituto de Investigación Sanitaria Biodonostia, Donostia, Spain 3 Instituto Nacional de Toxicología y Ciencias Forenses, Servicio de Biologia, Las Rozas, Spain 4 Instituto Vasco de Medicina Legal, Bioogia, Donostia, Spain 5 Hospital Universitario Donostia, Servicio Microbiologia, Donostia, Spain 6 University of Valencia-FISABIO, Joint Research Unit “Infection and Public Health”, Paterna, Spain

Molecular epidemiology and phylogenetic analyses have been used in the investigation of viral transmission cases in forensic contexts. Here, we present the methods and results of an analysis of a bacterial transmission case in an alleged child abuse case using complete genome sequences obtained by high-throughput sequencing (HTS) methods. We obtained genomes of Neisseria gonorrhoeae from the victim, the suspect, and 29 unrelated controls using Illumina NextSeq (150x2 bp, paired ends) technology. The analysis of the genomes revealed that the victim and suspect isolates had identical sequences in both the bacterial chromosome (2,173,861 bp) and the single plasmid (4,207 bp) present in them. One of the local controls was very similar (differing in only 2 SNPs) to the case sequences, but the remaining controls were very divergent. Additional cases of identity and very high similarity among controls were observed occasionally, pointing at recent transmission cases. These results were more discriminative than the previous molecular epidemiological analysis performed at the hospital’s Microbiology Service, since Multi-Locus Sequence Typing (MLST) could not distinguish between the suspect/victim and the control isolates, and Pulse Field Gel Electrophoresis (PFGE) was not able to distinguish between the suspect/victim and one of the local controls. These results lead us to conclude that complete bacterial genome sequences obtained with HTS technologies are a valuable tool for establishing recent transmission cases and can be used in forensic analyses.

78 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS O066 - WHOLE TRANSCRIPTOME ANALYSIS OF AGED BIOLOGICAL CRIME SCENE TRACES Andrea Salzmann1, Giancarlo Russo2, Sirisha Aluri2, Susanne Kreutzer2, Lars Snipen3, Cordula Haas1

1 Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland 2 Functional Genomics Centre Zurich FGCZ, University of Zurich/ETH Zurich, Zurich, Switzerland 3 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway

Current methods do not reveal information about the age of a trace or, more precisely, the time since deposition (TsD). From a criminalistic point of view, an accurate estimation of when the crime was committed enables to verify witnesses’ statements, limits the number of suspects and attests alibis. Furthermore, the assessment of TsD can help to identify the true relevance of a trace for the investigative process by linking its deposition to the time point of the crime. The goal of this study was to set up an RNAseq protocol to view potential TsD effects in the most common forensic stains (blood, saliva, semen, vaginal secretion, menstrual blood and skin) and to systematically analyse these effects in a time series experiment. In a test experiment several RNA-seq kits and protocols were tested and compared. Libraries were sequenced on an Illumina massively parallel sequencing platform. For the time series experiment we exposed samples to different environmental conditions, at room temperature in a dark and dry place, and exposed to the outside environment (sun, wind, etc.) but protected from rain. Total RNA was extracted at six different time points (0 day, 1 day, 7 days, 4 weeks, 6 months). We investigated human mRNA sequences and body fluid specific bacterial RNA signatures. Both approaches carried signals that made it possible to recognize the five body fluids. In addition, we also found systematic changes in the signals that correlate with TsD, indicating the RNAseq approach may be part of a solution to this difficult problem.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 79 O067 - PREVALENCE OF DNA IN VEHICLES: OPTIMIZING SAMPLING STRATEGY AND ACTIVITY LEVEL EVALUATION OF DNA TYPING RESULTS Tialda de Wolff1, Bart Aarts2, Margreet van den Berge2, Toni Boyko3,4, Matthijs Zuidberg5, Roland van Oorschot3,4, Bas Kokshoorn2

1 National Police of the Netherlands, Central Criminal Investigations Division, Driebergen, Netherlands 2 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands 3 La Trobe University, School of Molecular Sciences, Bundoora, Australia 4 Victoria Police Forensic Services Centre, Office of the Chief Forensic Scientist, Macleod, Australia 5 Netherlands Forensic Institute, Crime Scene Support Team, The Hague, Netherlands

There is an increasing demand on scientists in the field of forensic genetics to assess their source level findings in the context of a case. Evaluation of these findings given activity level propositions requires experimental data on the transfer, persistence, prevalence and recovery (TPPR) of DNA. One commonly encountered issue is the questioned driver of a vehicle. Did a person of interest (PoI) drive the vehicle during an incident, or was the PoI present as a passenger? Or did the PoI drive the vehicle, but at an earlier moment in time? We have studied the prevalence of DNA in vehicles that are operated by a single driver, either with or without a regular passenger, by sampling twenty standardized locations in the vehicles. We firstly identified high success rate sampling locations for intelligence purposes (e.g. identification through database searches). Secondly, the most informative sampling locations have been identified that could assist in assessing whether the PoI was either driver or passenger. Additionally we have introduced incidental drivers in vehicles that are regularly used by the owner. We have sampled DNA at three time intervals after use by an incidental driver, immediately after and at two different durations after being driven by a temporary user and subsequent continued use by the owner. These data inform the probabilities of persistence of DNA of both the owner of the vehicle, as well as that of the incidental driver. We explored the use of the obtained probabilities for TPPR of DNA to address propositions at activity level using real case examples. The results from this study allow further optimization of crime scene sample collection, as well as activity level assessment of findings in a case based on probabilities for TPPR of DNA in vehicles.

80 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Posters

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 81 FORENSIC GENETICS AND GENOMICS OF HUMAN AND NON‑HUMAN BIOLOGICAL SAMPLES

P001 - A CASE OF MOTHER-DAUGHTER RELATIONSHIP WITH 5 LOCI INCONSISTENT WITH HEREDITARY LAW IN 56 AUTOSOMAL STR MARKERS Ying Liu1,2, Jingjing Zhang3, Jiajia Liu1,4, Jiangwei Yan5, Yacheng Yacheng Liu6

1 Di’an Institute of Forensic Sciences, Di’an Institute of Forensic Sciences, Beijing, China 2 Forensic Laboratory, Gansu Di’an Tongxiang Medical Laboratory, Lanzhou, China 3 Beijing Huayan Judicial Authentication Institute, Beijing Huayan Judicial Authentication Institute, Beijing, China 4 Shanghai Di’an Forensic Sciences CO.- LTD, Shanghai Di’an Forensic Sciences CO.- LTD, Shanghai, China 5 School of Forensic Medicine, Shanxi Medical University, Taiyuan, China 6 Beijing Tongda Shoucheng Institute of Forensic Science, Beijing Tongda Shoucheng Institute of Forensic Science, Beijing, China

Paternity testing was investigated between mother and daughter (mother 47 years old & daughter 25 years old). The total of 56 autosomal STR loci, 86 autosomal SNPs, 19 X-STRs and the whole mitochondrial genome were detected by capillary electrophoresis (CE) and Next-generation sequencing (NGS). Five out of 56 autosomal STR loci were found to be inconsistent with mendelian inheritance. (Five loci that do not conform to the hereditary law are distributed on five chromosomes, among which four loci differ by one repeat unit and two repeat units on D21S1270 locus.) 86 autosomal SNPs and 19 X-chromosome STR loci conform to the genetic development. The whole mitochondrial genome sequence of the two person is identical. According to the test results, we set Hp hypothesis that there is a mother-daughter relationship between two persons.

HD is an aunt-nephew relationship between two persons. The LR values of autosomal STR, autosomal SNP and X-STR are 4.38*10-6, 1.17*106 and 9.78 respectively. Considering comprehensively, it is considered that there is a mother-daughter relationship between them.

82 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P002 - A COMPARISON OF ENDOGENOUS AND EXOGENOUS REFERENCE MARKER AS RELEVANT FOR ACCURATE POST‑MORTEM INTERVAL ESTIMATION Sonja Uerlings1, Burkhard Madea1, Melanie Grabmüller1

1 Institute of Legal Medicine, University of Bonn, Bonn, Germany

The Post-Mortem Interval (PMI), also known as time since death, is of relevance for forensic routine casework and serves primarily as the basis for the determination of the time that has passed since the time of death. The existing, “classical” approaches are characterised by certain limitations which is why they only work for the early Post-Mortem Interval. Therefore, it was tried to establish a new method for the PMI-determination by means of a RNA-based gene expression analysis and normalisation by means of endogenous or exogenous reference marker. The present study deals with the assessment of various molecular mRNA-marker (e.g. HIF1A & HSPA1B) and so called circadian mRNA-markers in different kinds of post-mortem human specimens (e.g. heart & skeletal muscle tissue) concerning their ability to use them for the estimation of the time since death. Moreover, a comparison between carefully selected endogenous and one exogenous reference marker for the normalisation of the gene expression data as relevant for accurate PMI estimation is drawn. In addition, it should be clarified to what extent the gene expression is influenced by factors, such as age or gender of the deceased. The evaluation of the samples with exactly known PMI (death – autopsy of the dead body) showed that the used approach (quantitative real-time polymerase chain reaction) is meth- odologically useful but the tested exogenous reference marker was not suitable for sufficient data normalisation. The tendency of a rhythmic expression pattern for the circadian genes was demonstrated for the blood samples. A significant correlation between the expression level and the PMI as well as the other factors of influence did not exist when the exogenous reference marker was used for data normalisation.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 83 P003 - A CONTINUOUS SAMPLING TECHNIQUE FOR THE STUDY OF SOFT TISSUE DECOMPOSITION Samara Garrett-Rickman1, Maiken Ueland1, Dennis McNevin1, Shari Forbes2

1 University of Technology Sydney, Centre for Foresic Science, Sydney, Australia 2 Université du Québec à Trois-Rivières, Département de chimie- biochimie et physique, Trois-Rivières, Canada

Studies relating to human decomposition are integral to creating a reliable foundation for forensic taphonomy. Experimental data will allow for a greater understanding of the influencing variables, and ultimately assist in creating a reliable method for post-mortem interval (PMI) determination. The ability to continuously sample from whole human cadavers is necessary for a holistic view of the taphonomic changes occurring at each stage of decomposition. Due to experimental and ethical constraints, there is limited research into the relationship between soft tissue samples and PMI. This study aimed to develop a method for the continuous sampling of skeletal muscle tissue from whole human cadavers placed at the Australian Facility for Taphonomic Experimental Research (AFTER), through the evaluation of conventional and new sampling techniques, and explored the potential for in-field application.

84 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P004 - A COUPLE OF CHIMERIC TWINS GENERATED FROM A POSSIBLE INTRAUTERINE CELL EXCHANGE Dante Corradi1, Laura Locarno1, Miguel Marino1

1 Public Ministry of Mendoza, Registro Provincial de Huellas Genéticas Digitalizadas, Mendoza, Argentina

Chimerism is the presence of two or more genetically distinct cell lines (distinct zygote lines) in an organism. The presence of blood Chimerism in human twins is a rare event and was first described by Dunsford et al. (1953) who discovered a mixture of two blood types in a healthy donor. The only way some types of chimeras can be distinguished from non-chimeras is trough extensive DNA testing. In 2017 in the context of a sex assault, a blood sample came to our Forensic Genetics Laboratory. When analyzed with an STR kit and the results were confirmed, the obtained profile surprised us. A male mixed profile was observed, with up to 4 alleles per marker and showing imbalances in some of them. After evaluating and confirming that it was not a contamination this arouse our interest in deepen the study of this sample. We were able to find out that the individual to whom the sample belonged had a twin brother. Thus, after obtaining the consent of both individuals more samples were taken for further studies. Blood, hair and nails of both were analyzed with different STR, Y and X chromosome markers. In hair samples we obtained single males profiles, but in blood samples these profiles were mixed together in different proportions. Those alleles present in the hair samples of one subject where the minor contributor in the blood profile of his sibling and vice versa. We present a brief summary of the case context and explain the results of the battery of DNA markers that we used in this case.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 85 P005 - A FAMILY REUNITED AFTER APPROXIMATELY 5000 YEARS Elena Pilli1,2, Stefania Vai1, Anna Anselmo2, Andrea Berti2, David Caramelli1

1 University of Florence, Department of Biology, Firenze, Italy 2 Molecular Biology and Genetics Unit, Carabinieri Scientific Investigation Department, Roma, Italy

The ability to recover and analyze DNA from highly degraded skeletal remains represents one of the most significant challenges for molecular anthropologists and forensic experts. Foren- sic genetic investigation is conducted by the analysis of STRs, and/or D-loop control region of mitochondrial DNA (mtDNA). Recently, development of Next Generation Sequencing (NGS) offered the possibility to obtain information even from highly degraded samples that could not be analyzed with routinely forensic methods. The aim of this study was to analyze a group of skeletal remains applying advanced molecular procedures based on NGS and target enrichment by hybridization. Different bone/teeth samples dated from 2500–3500 BCE were recovered by a multiple secondary burial. Most of skeletal material was fragmented and mixed, and belonged to at least 23 different individuals. 597,573 nuclear SNPs and whole mitochondrial genome analysis was performed on 17 samples from 17 different individuals. Alleged age and sex estimation was initially assessed by anthropological examination. Whole genome mtDNA data highlighted four individuals had the same haplotype, suggesting a possible matrilineal kinship between them. SNPs analysis confirmed the kinship relations between three out of four individuals (two samples belonged to the same individual), showing the presence of a mum with her two children. In addition, nuclear DNA analysis permitted to discover between the skeletal remains also the father of this family. In conclusion, NGS technology, associated to target enrichment capture, permitted to verify the minimum number of individuals, assess sex, and kinship relations and to identify three samples carrying a lower misincorporation pattern as result of a burial reuse in the recent times.

86 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P006 - A METHOD FOR SCREENING MICROHAPLOTYPE LOCI FROM GENOME-WIDE POPULATION DATA Peng Chen1, Zheng Li1, Yan Pu2, Jiawen Yang1, Weibo Liang3, Lin Zhang3, Feng Chen1

1 Nanjing medical university, Forensic medicine, Nanjing, China 2 Southeast university, School of medicine, Nanjing, China 3 Sichuan university, Forensic medicine, Chengdu, China

Microhaplotype have been proved to be a very potent genetic marker in forensics. Increasing researches have suggested that microhaplotype could be applied in individual identification, mixture analysis, ancestry inference and kinship analysis. Since there are over 80 million SNPs that could further generate microhaplotypes, they are abundant in the human genome. Dif- ferent microhaplotype loci can be used in different forensic purposes. For instance, microhaplotypes with high effective number of alleles (Ae) are suitable for DNA mixture analysis, while microhaplotypes with high informativeness for ancestry inference (In) can be used to distinguish populations with different ancestor. However, how to extract enough microhaplotypes from genome-wide data is still an unsolved issue. In the present study, we introduce a rapid novel method for screening microhaplotype loci from genome-wide population data. Since 1000 genome project provides very divergent wide-genome data including 2504 individuals from 26 populations, we therefore used the data from the 1000G database to test our screening strategy. Firstly, we sorted the SNPs based on their chromosome information to discriminate different microhaplotypes according to their physical position. We next calculated the haplotype frequencies based on the minor allele frequencies of each individual SNP in the microhaplotypes. Finally, microhaplotypes can be sorted by the number and frequency of the alleles.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 87 P007 - A NOVEL SEMI-AUTOMATED DIRECT LYSIS METHOD FOR DNA RECOVERY FROM LIVE AND SPENT 9 MM AMMUNITION Zuhaib Subhani1, Kiera Coleman2, David Moore1, Tim Clayton3, Melanie K Brown2

1 Eurofins Forensic Services, DNA Research and Development, Teddington, United Kingdom 2 Teesside University, School of Science - Engineering & Design, Middlesbrough, United Kingdom 3 Eurofins Forensic Services, Casework - Examination and Reporting, Wakefield, United Kingdom

Recovery of cellular material and DNA from ammunition is a potentially valuable process capable of providing probative evidence of criminal use of firearms. However, DNA profiling success rates on ammunition are low, and consequently much of the ammunition recovered from crime scenes is never submitted for DNA analysis. There is also a common assumption that DNA from fired ammunition is likely to have deteriorated following discharge. In this study, DNA recovery and subsequent STR profiling was conducted on live and spent 9 mm ammunition, handled by known donors prior to loading. Two methods were compared; the commonly-used double swabbing technique and a novel semi-automated direct lysis method using AutoLys tubes (Hamilton). The direct lysis method involved placing 9 mm cartridges into an AutoLys tube and submerging the cartridge in lysis buffer prior to purification. Lysate was recovered by centrifugation facilitated by the AutoLys tube design. It was found that the direct lysis method recovered significantly more DNA and yielded correspondingly improved STR profiles than the double swabbing technique. It was also shown that DNA could be recovered and profiled using the direct lysis method on both live and spent 9 mm cartridges. These results demonstrate that DNA suitable for STR analysis can be recovered from spent ammunition with only slightly reduced yields compared to live ammunition. In many cases the last handler of the ammunition was a major contributor to the recovered DNA. Results will also be presented on the effects of the direct lysis reagents on ballistic marks and on DNA recovery from different 9 mm ammunition types.

88 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P008 - A STEPWISE STRATEGY TO DISTINGUISH MENSTRUAL BLOOD FROM PERIPHERAL BLOOD BY FISHER’S DISCRIMINANT FUNCTION Anquan Ji1, He Hongxia1, Zhao Yixia1, Hu Sheng1, Ye Jian1, Liu Yao1, Sun Qifan1

1 institute of forensic science, Innovation and Development Research, Beijing, China

Abstract: Blood samples are the most common and important biological samples at the crime scene, and blood samples there belonging to peripheral blood or menstrual blood are crucial for solving criminal cases. MicroRNA (miRNA) is an important biological molecule with strong tissue specificity that can be used in forensic fields to identify the tissue properties of body fluid samples. In this research, the relative expression levels of four kinds of miRNAs (miR-451, miR-205, miR-214, and miR-203) were analysed by real-time PCR, with 200 samples from 5 different kinds of body fluids, including two kinds of blood samples (peripheral blood and menstrual blood) and three kinds of non-blood samples (saliva, semen and vaginal secretions). Then, a strategy for identifying menstrual and peripheral blood based on Fisher’s discriminant function and the relative expression of multiple miRNAs was established. Two sets of functions were used: Z1 and Z2 were used to distinguish blood samples from non-blood samples, and Y1 and Y2 were used to distinguish peripheral blood from menstrual blood. A 100 % accuracy rate was achieved when 50 test samples were used. Ten samples were used to test the sensitivity of the method, and it is recommended that 10 ng or more of total RNA from peripheral blood samples and 10 pg or more of total RNA from menstrual blood samples are sufficient for conducting this method. This result provides a scientific reference scheme for solving the difficult forensic problem of distinguishing menstrual blood from peripheral blood. Key words: body fluid identification, MicroRNA, peripheral blood, menstrual blood, Fisher’s discriminant function

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 89 P009 - A STUDY OF HUMAN REMAINS USING MPS METHODS Eida Almohammed1, Dragana Zgonjanin2, Saleh Alsaadi1, Fatma Al-Ali1, Hadi Sibte3

1 Ministry of Interior Qatar, Qatar Forensic Laboratory, Doha, Qatar 2 University of Novi Sad, Faculty of Medicine, Novi Sad, Serbia 3 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom

DNA analysis from human remains is of immense relevance in missing persons identification and disaster victim identification (DVI). DNA degrades gradually in hard tissues, such as bones and teeth under a high temperature, humidity, pH, geochemical properties of the soil, the presence of microorganisms and all other factors that affect the preservation of DNA in skeletal remains. Recent advances in massively parallel sequencing (MPS, provides some advantages over previously used technologies to analyse DNA from human remains, particularly ancient. The aim of this study was to investigate if we could use the Illumina ForenSeq DNA signature kit to genotype autosomal STRs, X and Y STRs as well as SNPs in bone samples for use in disaster victim identification. The beta-version of the ForenSeq kit was used to genotype 32 bone samples from Serbia lab that previously profiled using GlobalFiler amplification kit using 3500 capillary electrophoresis (CE). A total number of 86 samples were typed on 3500 CE and 32 were selected for NGS work. Results of Run on FGx-MiSeq sequencing showed a cluster density of 866 K/mm2. A total 95 % of clusters generated run passed filters. The results of the samples will be shown between the CE and NGS results. The beta-test worked rather well for a beta-test using one independent run. Ancestry, Identity and phenotypic SNPs had been typed in the degraded samples. The CE GlobalFiler data -STR markers and the Illumina ForenSeq DNA signature kit showed remarkable results for concordance.

90 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P010 - A STUDY OF SKELETAL REMAINS USING GLOBALFILER™ KIT Eida Almohammed1, Dragana Zgonjanin2, Hadi Sibte3

1 Ministry of Interior of Qatar state, Qatar Forensic Laboratory, Doha, Qatar 2 University of Novi Sad, Faculty of Medicine, Novi Sad, Serbia 3 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom

DNA analysis from human remains is of immense relevance in missing persons identification and disaster victim identification (DVI). DNA degrades gradually in hard tissues, such as bones and teeth under a high temperature, humidity, pH, geochemical properties of the soil, the presence of microorganisms and all other factors that affect the preservation of DNA in skeletal remains. The GlobalFiler™ kit simultaneously amplifies and detects 21 autosomal loci including CODIS extended set of STR loci. The kits was designed to combine all 21 autosomal STR loci along with a novel male speciﬁc Y insertion/deletion marker the sex-determining marker, amelogenin. Thus, GlobalFiler™ combines the CODIS extended set of loci and includes seven autosomal STR loci from the expanded European Standard Set of Loci (ESSL). The kit also includes the highly discriminating SE33 locus. The aim of this study was to profile the old skeleton remains using GlobalFilerTM for use in disaster victim identification that previously profiled using Identifiler Plus kit using 3500 capillary electrophoresis (CE). This study investigated the success rate of short tandem repeat (STR) typing from different types of bone samples observed a higher STR success rate using GlobalFiler™ PCR kit, which contains more than 10 loci with a maximum size of 200 bp. Thus, GlobalFiler™ could potentially be the optimum amplification choice for the limited amounts of DNA obtained from challenged bone samples. The performance of the GlobalFiler™ kit and the profile quality was examined for the degraded bone samples to determine if it could generate more robust genetic information.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 91 P011 - A USEFUL SOLUTION IN CASES OF HUMAN IDENTIFICATION Eliza Michalak1, Monica Abreu-Glowacka1, Czesław Żaba1

1 Poznan University of Medical Sciences, Department of Forensic Medicine, Poznan, Poland

In recent years, our forensic genetics laboratory carried out a study on human identification, especially when human bodies were in high level of decomposition. For this study 32 fragments of humerus shafts (n=18) and femoral shafts (n=16), 23 teeth and 11 phalanges were studied. Additionaly, nails were also collected for each case of human identification. The bone material, teeth and nails were cleaned and properly prepared for DNA isolation and DNA amplification using the STR-PCR method. Out of 66 cases of human identification in 26, a full genetic profile was obtained from examined bone material (15 cases) and teeth (11 cases), which constitutes 39 % of all performed tests, while the genetics analysis of nails in the same cases, provided a full genetic profile in 63 cases, which accounts for 95 % of the conducted research. Currently in our laboratory, nails are collected, in addition to bone material, in each cases of human identification, when body is in a state of high decomposition. It seems that it is a useful solution in forensic human identification.

92 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P012 - ALLELIC DIVERSITY AND FORENSIC ESTIMATIONS OF THE BEIJING HANS: COMPARATIVE DATA ON SEQUENCE‑BASED AND LENGTH-BASED STRs Kelai Kang1,2, Chen Qingfeng1,2, Zhang Chi1,2, Ji Anquan1,2, Wang Le1,2

1 National Engineering Laboratory for Forensic Science, Institute of Forensic Science, beijing, China 2 Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, beijing, China

Short tandem repeat (STR) profiling is routinely used in forensic genetics. At present, the analysis of STRs is mainly performed by capillary electrophoresis (CE). However, due to limitations of the CE method, STR genotyping can only be limited to length polymorphism. Next generation sequencing (NGS) is capable of providing full resolution STR data at the sequence variation level, which could significantly improve the individual identification capability of forensic STR loci. Here we present sequence-based STR data for the Beijing Han population. 291 individuals were sequenced for 23 commonly used forensic STRs using the SeqTypeR24 CASE kit on Ion PGM platform. 233 length-based alleles and 355 sequence-based alleles were observed, including 22 novel core repeat sequences. The sequence-based matching probability and power of discrimination were superior to the length-based numbers for 16 loci bearing micro-variant alleles. Combined matching probability reached 8.2×10-29 for 23 STR loci at the sequence level, which is 2 orders of magnitude higher than the parameter at length level, providing data base for sequence-based STR casework applications.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 93 P013 - AN APPROACH TO EXTRACTING USEFUL SEQUENCE INFORMATION FROM COMPLEX MIXTURES OF HUMAN DNA Leigh Burgoyne1, Lin Y.Koh1, David Catcheside1

1 Flinders university, sci&eng, Bedford Park, Australia

Metagenome sequencing is theoretically capable of resolving individuals present in samples having multiple contributors. We are investigating ways of achieving this using economic single-primer high-cycle-number amplification methods. DNA from a single human and a mixture of DNA from a very large number of individuals was amplified separately using a single primer and subjected to illumina Nextera 150bp paired-end sequencing. High informational quality sequences were extracted and contiged. In Blastn searches on NCBI, the contigs were always attributable to the human genome with highest quality matches to humans with the expected poorer matches to orthologs in the human genome and lesser matches to paralogs in other primates. In the case of the large number of remnants of transposable elements present, sampling showed the sequences between the inverted repeats were from human DNA whist the inverted repeats were found in a wide diversity of biota. The diversity of transposon remnants may be forensically useful. The sequences used in this analysis will be available for download at the meeting.

94 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P014 - AN ENHANCED DNA AMPLIFICATION METHOD TO DETECT THE SPECIES ORIGINS OF THE ROOTLESS HAIR SHAFTS Chong Chen1,2, Yuan Lin2, Ruiyang Tao2,3, Jjiashuo Zhang2,4, Jingyi Zhang2,4, Zihao Yang5, Chengtao Li2,3, Suhua Zhang2

1 College of Medicine and Forensics, Xi’an Jiaotong University Health Science Center, Xi’an, China 2 Academy of Forensic Science- Ministry of Justice, Forensic Genetics, Shanghai, China 3 Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine- Sichuan University, Chengdu, China 4 Department of Forensic Science, Medical School of Soochow University, Suzhou, China 5 Department of Forensic Medicine, School of Basic Medical Science- Wenzhou Medical University, Wenzhou, China

Hair is a common biological sample at crime scene. Determining the biological origins of hairs is important for case investigation. However, the DNA extraction of rootless hair shafts are quite difficult. In this study, an enhanced DNA amplification methodology is revealed, in combination with a self-developed system of 5×PCR Reaction Master Mix IV, to directly amplify the mtDNA 12S ribosomal RNA (12S rRNA) gene from rootless hair shafts collected from 12 common species (human, chicken, duck, cattle, sheep, dog, mouse, pig, horse, cat, rabbit and pigeon) without DNA extraction. The amplicons were subsequently sequenced on Pyromark Q48 (QIAGEN, Hilden, Germany). The results illustrated that the direct amplification system could efficiently amplify the targeted mtDNA genes and pyrosequencing technology could further readily differentiate the specimen origin of the rootless hair shafts. The data analysis of the 12S rRNA was equipped to an automatic analysis module. This study provides a new solution for species identification of rootless hair shafts.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 95 P015 - AN UNUSUAL KINSHIP CASE FROM THE SPANISH CIVIL WAR (1936–1939): ANCIENT VERSUS CRITICAL SAMPLE’S INVESTIGATION Cláudia Gomes1,2, Manuel Fondevila3, Concepción Magaña-Loarte4, Juan Fernández5, José Fernández-Serrano5, Sara Palomo-Díez1,2, Carlos Baeza-Richer1,2, Ana María López-Parra1,2, Eduardo Arroyo-Pardo1,2

1 Facultad de Medicina Universidad Complutense de Madrid, Legal Medicine, Psychiatry and Pathology, Madrid, Spain 2 Instituto de Investigación Sanitaria del Hospital Clínico San Carlos IdISSC, Grupo de Ciencias Forenses: Genética y Toxicología forenses, Madrid, Spain 3 Institute of Forensic Sciences INCIFOR- Forensic Genetics Service, Forensic Genetics Service, Santiago de Compostela, Spain 4 Laboratory of Forensic Anthropology, Forensic Anatomical Institute, Madrid, Spain 5 Criminalistics Service- Civil Guard, Laboratory of the Biology Department, Madrid, Spain

An unusual kinship case arrived to our laboratory, having as main objective to perform a genetic analysis between a living presumable niece and a human remain –eight bones from a hand. The bone samples probably belonged to a cleric executed in Almeria (Spain), during the Span- ish Civil War (1936–1939), where, after the execution, the cadaver was thrown to a dry well and covered with lime. Later, the cadaver was transferred to a local cemetery, where due to a flood, was then relocated in another sanctuary. It was at this moment when the hand was separated from the other remains, and kept under the control of the family for decades. After an anthropological study, the three best specimens from the eight available bones were selected to perform the genetic analysis. After the DNA extraction, a mitochondrial DNA analysis was accomplished to test the alleged maternal uncle-niece relationship. The results confirmed the bones degradation, since each sample produced different mitochondrial sequences, being impossible to obtain a consensus profile. It was impossible to confirm any kinship between the incomplete hand and the living person, or to conclude if the bones were part of the same hand. Although our study focused on samples with less than one 100 years old, similar investigations have been previously successfully achieved in our laboratory on very degraded samples, for example, from the Chalcolithic Period (Palomo-Díez et., 2015). In that study, the obtained results allowed, not only to stablish biological kinships, but also to associate different human remains. It is therefore crucial to distinguish between both concepts – ancient versuscritical (samples). The elapsed time since the sample deposition may not influence in the results as much as the conservation conditions.

96 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P016 - ANALYSIS AND INTERPRETATION OF MIXTURE DNA USING AS‐PCR OF mtDNA Bao-Jie Wang1, Feng-ling Xu1, Mei Ding1, Jun Yao1, Xue Wu1, Jia-xin Xing1, Jin-feng Xuan1, Xi Xia1

1 China Medical University, School of Forensic Medicine, Shenyang, China

In forensic medicine, mixed stains usually include body fluids, secretions of two or more individuals. The application of STR profiling of single cell capture or mixed samples requires that the existence of intact nucleated cells or the appropriate proportion of different components in the sample, respectively. Trace components cannot be genotyped, and the analysis of mixture from more than two individuals is even more difficult. There are SNPs located in the mtDNA hypervariable region and its flanking sequences. Accord- ing to the differences of SNPs between individuals, allele-specific PCR (AS-PCR) may be used to obtain the individual DNA fragments containing mtDNA hypervariable regions in mixed stains. There are two assumption in the application of our method. One assumption is that when the suspect is known, the AS-PCR can be designed according to the SNPs in the mtDNA hypervariable and flanking regions. The suspect cannot be excluded if the amplified sequence is consistent with the suspect’s. Otherwise, the suspect can be excluded. The other assumption is without the suspect. We can obtain the individual’s sequence of the high component and then perform the multiple AS-PCR according to specific SNP. If the low component’s fragment can be amplified, it is possible from the potential suspect. Our method is easy to conduct and can exclude the unrelated individuals or provide the suspect’s information, especially for the individual with the low component. To some degree, it can solve the analysis the mixture of multiple individuals. However, it is invalid for the samples of the same maternal line and the personal identification cannot be achieved. Acknowledgments: Our study is supported by National Natural Science Foundation of China (NO. 81671872).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 97 P017 - ANALYSIS OF 16S rDNA IN SOIL BACTERIAL BY T-RFLP AND ITS APPLICATION IN LEGAL MEDICINE Xudong Wang1

1 Southwest University of Political Science and Law, Forensic Science Engineering Research Center of Universities in Chongqing, Chongqing, China

Objective: To evaluate the validation and application of T-RFLP method in differentiating the soil from varying plots in China. Methods: Soil samples from ChongQing and JiLin Province were treated with PowerSoil® DNA extraction kit. PCR was done to amplify the hyper-diversity 16S rDNA gene with the FAM-labeled primers. The PCR product were cut with four different restriction enzymes MspI, RsaI, HaeIII and AluIrespectively. The digestion fragments were performed electrophoretic separation on a 3130 Genetic Analyzer. Statistics with PCA and AMMI were done to observe the diversity. Results: The T-RFLP with HaeIII and MspI restriction enzyme could discriminate the soil samples from ChongQing and Jilin Province. However, RsaI and AluI enzyme failed. Conclusion: The polymorphism of soil microbial diversity depends on the varying of geological location and soil nature. T-RFLP with appropriate enzyme offers a promising method to discriminate soil samples from particular site, which may serve as an effective approach for soil diversity.

98 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P018 - ANALYSIS OF DNA TRANSFER TO FIREARMS UNDER REALISTIC CONDITIONS CONSIDERING RELEVANT ALTERNATIVE HANDLING SCENARIOS Annica Gosch1, Jan Euteneuer1, Johanna Preuß-Wössner1, Cornelius Courts1

1 University Medical Center Schleswig-Holstein, Institute of Forensic Medicine, Kiel, Germany

The recovery of skin contact traces from firearms is routinely performed in forensic DNA laboratories, aiming at identifying the person who fired the weapon in gun-related crimes. However, aspects like DNA deposit, transfer, persistence and recovery need to be considered when interpreting trace DNA profiles recovered from assumedly touched items. Using DNA-TrAC, a searchable database on DNA transfer studies [1], we confirmed a lack of knowledge regarding the constitution and the composition of trace DNA profiles on firearms. In the present study, we thus evaluated the effect of alternative handling scenarios on the amount of DNA recovered from various surfaces of pistols and revolvers and the resulting profiles’ composition. Background DNA was deposited on the firearms in a realistic “owner” scenario in which participants handled and stored the firearm in their own living environment prior to the actual “shooting” scenarios: a short handling consisting of pulling the trigger and disposing of the weapon, a longer and more intense handling and a short handling followed by wiping off fingerprints with a fabric towel. To enable a direct comparison of casework-relevant alternative propositions, a fourth scenario was included in which the owner himself acted as the shooter. The presented data will provide useful information on factors impacting the composition of biological traces on firearms and their potential to differentiate between alternative activity level propositions which will support evidence-based reconstruction of gun-related crimes. [1] A. Gosch and C. Courts, “On DNA transfer: The lack and difficulty of systematic research and how to do it better,” Forensic Sci. Int. Genet., vol. 40, pp. 24–36, 2019.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 99 P019 - ANALYSIS OF FULL AND HALF SIBLINGS USING A STR, INDEL AND SNP MARKERS COMBINED SYSTEM Xiling Liu1,2,#, Zhenmin Zhao2#, Qiannan Xu2, Jiayi Zhang2, Chengtao Li2

1 Academy of Forensic Science, Shanghai Key Laboratory of Forensic Medicine, Shanghai, China 2 Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, P.R. China, Shanghai, 200063, P.R. China

Analysis of full and half siblings using a STR, InDel and SNP markers combined system (P) Paternity and first-degree relationship, like full-siblings, can be identified by current commonly used forensic short tandem repeats (STRs). However, discrimination of second-degree and more distant relationships, which is frequently appeared in judicial practice, remains challenging. Previous studies have shown that single nucleotide polymorphisms (SNPs) contain a large amount of genetic information. The strategy of a STR and SNP combined system was suggested to reduce the costs and procedures of genotyping, and show great value in kinship testing. In this study, to test how many markers are sufficient to perform the kinship testing for siblings, 60 STRs, 30 insertions and deletions (InDels), as well as 472 SNPs were firstly collected. The specificity to distinguish full siblings was no less than 99.99 % under FDR = 0 when at least 57 out of the 60 STRs were used. The specificity to distinguish half siblings was 99.93 % under FDR = 0 when all the markers were used. In total, the combined marker system showed promising for solving the problem of sibling relationship identification. # Contributed equally * Corresponding authors: Xiling Liu (e-mail: [email protected], phone number: +862052351327) and Chengtao Li (e-mail: [email protected], phone number: +862052351327).

100 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P020 - ANALYSIS OF SKELETAL REMAINS EXHUMED FROM A “DRY WATER WELL” USED AS A SECONDARY CLANDESTINE BURIAL IN ARGENTINA Maria Laura Catelli1, Magdalena Romero1, Andrea Rocha1, Martina Rotondo1, Carlos Vullo1

1 Argentine Forensic Anthropology Team EAAF, Forensic Genetic Laboratory LGF-EAAF, Cordoba, Argentina

The military dictatorship that took power in Argentina from 1976 to 1983, brought enforced disappearance and death to thousands of people in the country. “Pozo de Vargas” is a 3.5 meters wide and 60 meters deep dry water well used as a secondary clandestine burial site in Tucumán, Argentina. The site contained commingled remains as well as partially articulated body parts. Since 2011, EAAF has been continuously processing post mortem (PM) samples exhumed from the well to achieve the intra-skeletal re-association and the identification of the bodies or body parts. 982 skeletal remains, in varying states of preservation, were analyzed for STR typing using commercial available kits. Different bone elements were exhumed for intra-skeletal re-associations (PM-PM genetic comparisons): teeth, femur and tibia represented more than 60 % of sampling and almost all of them were successfully re-associated; spongy bones as vertebrae, ribs and foot bones yielded worse results although almost 80 % of them were also re-associated. 886 skeletal remains (90 %) were successfully re-asociated into 147 unique STR profiles; it means at least 147 different individuals were exhumed from the water well. After re-association, unique STR profiles obtained from the skeletal remains were compared to ante mortem (AM) family reference database in order to identify victims (AM-PM genetic comparisons), being possible to restore 113 victims to their families. Another challenge of this identification project was the presence of biologically related victims inside the well.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 101 P021 - APPLICATION OF 13 LOCI STR MULTIPLEX FOR CANNABIS SATIVA GENOTYPING Matteo Fabbri1, Paolo Frisoni1, Matteo Marti1, Anna Talarico1, Omar Bonato1, Mauro Coppone1, Elena Lucenti1, Rosa Maria Gaudio1, Margherita Neri1

1 University of Ferrara, Department of Morphology- Surgery and Experimental Medicine - Institute of Legal Medicine, Ferrara, Italy

As human DNA, plant DNA have a great potential as a useful genetic evidence tool in forensic analysis and investigations. The most common system used for discrimination between individuals in forensic genetics, short tandem repeat (STR) markers, have recently been developed for cannabis sativa. As in the other countries of the world, in Italy marijuana represents the most commonly used illicit substance and it has become a highly trafficked drug to and within Italy by organized crime. Consequently, a novel method needs to be establish for individualization and origin determination of cannabis samples, in order to establish a certain link between cannabis cases. Relying on the topics previously described, this study had the aim to provide a STR marker set having a good power of discrimination to help investigative authorities to trace back trade routes and to link plants to a specific dealer or crime scene. Our work describes an application of a STR multiplex systems for 13 loci, previously developed and validated in our laboratory following international guidelines, on 4 cannabis specimens seized by Anti-Narcotic Italian Police. STR loci were chosen from the set of publicly available STRs for cannabis sativa and among the highly polymorphic ones. Gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) analysis revealed and confirmed four comparable molecular spectrums, approximatively descriptive of the same plant/seizure origin. Due to these uncertain results, investigative authorities approved the genetic analysis on the same samples. DNA profiling allowed to identify the same genetic profile and consequently the same origin and individualization of the specimens analyzed.

102 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P022 - APPLICATION OF DROPLET DIGITAL PCR TO SIMULTANEOUS QUANTIFICATION OF NUCLEAR DNA AND MITOCHONDRIAL DNA Tsukasa Ohuchi1, Xueting Guan1, Yui Igari1, Kiyotaka Usui1, Funayama Masato1

1 Tohoku University, Forensic Medicine, Sendai City, Japan

Background and aims: DNA typing for human identification is usually performed by short tandem repeat (STR) analysis targeting nuclear DNA (nDNA). However, when samples are severely degraded, mitochondrial DNA (mtDNA) analysis is carried out. To efficiently determine which analysis is feasible to be applied to various forensic samples, Timken et al. reported simultaneous quantification of nDNA and mtDNA using real-time PCR [1]. However, more simpler methods are required because real-time PCR has a drawback of requiring a standard curve for each target. Recently, droplet digital PCR (ddPCR) has emerged as a promising method because it does not require a standard curve. Therefore, we investigated to apply ddPCR for simultaneous quantification of nDNA and mtDNA. Materials and methods: Primer sets and TaqManTM probes were prepared as described by Timken et al [1]. K562 DNA (Promega) was used as a test sample. Using QX200 Droplet Digital PCR system (Bio-Rad), the optimal conditions for ddPCR were examined. Results: nDNA and mtDNA were tested separately and the optimal annealing / extension temperature was determined to be 55 °C. In addition, for mtDNA, the concentrations of primers and probe were increased because the positive signal was low. The simultaneous quantification system was tested under the optimized conditions and it was verified that both targets were quantified in the same well. Conclusions: In forensic DNA investigation, it is important to check the quality of extracted DNA to decide the subsequent workflow. This study suggested the applicability of ddPCR in a forensic usage. To improve reliability, further research is needed, such as a comparison of this approach with real-time PCR. Reference: [1] Timken MD et al. J Forensic Sci. 2005 ; 50 : 1044–1060.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 103 P023 - APPLICATION OF METAPOPULATION ANALYSIS OF THE 16S RNA REGION IN CASES OF SEXUAL CRIMES Jakub Czarny1, Paweł Cyplik2, Joanna Idkowiak1, Monika Antczak1, Aleksandra Górka1, Łukasz Kaczorowski1, Bartosz Hornik1, Agnieszka Piotrowska-Cyplik3, Jolanta Powierska-Czarny1, Magdalena Chrostowska1

1 Institut of Forensic Genetics, Department of Forensic Genetics, Bydgoszcz, Poland 2 Poznan University of Life Sciences, Department of Biotechnology and Food Microbiology, Poznań, Poland 3 Poznan University of Life Sciences, Institute of Food Technology of Plant Origin-, Poznań, Poland

In cases of sexual crimes in which semen is not ejaculated, it may be necessary to identify the origin of the male genetic material as preputial specimen and the female genetic material as vaginal specimen. We present the use of metapopulation analysis of region IV of the 16S RNA gene in one of the sexual offense cases. In case of a 60-year-old man suspected of penetrating a vagina, paper towels were secured, on which PSA and Semenogelin were not established, and a mixture of genetic material of the victim and the suspect was found. The swabs from the vagina and the mouth of the victim, swabs from the prepuce and mouth of the suspect, and biological traces revealed on the paper towel were subjected to metapopulation studies. Region IV of bacterial 16S RNA gene was amplified using universal primers 515F and 806R: containing reverse complement of 3’ Illumina adapter, golay barcode, reverse primer pad, reverse primer linker and reverse primer. The libraries were constructed from amplicons using NEBNext® DNA Library Prep Master Mix Set for Illumina (New England Biolabs UK). Sequencing was conducted using an Illumina MiSeq (Illumina, USA) using paired-end (2x250) MiSeq Reagent Kits v2 (Illumina, USA). The sequencing data was processed using CLC Genomic Workbench 8.5 and CLC Microbial Ge- nomics Module 1.2. (Qiagen, USA). No bacteria were found in the secured traces on the paper towel and in the mouth of the victim or the suspect, while the components of the bacterial microflora found in the victim’s vagina and under the prepuce of the suspect were identified. The obtained results indicate that metapopulation analyses within the regions of the 16S RNA gene may be a useful forensic tool.

104 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P024 - APPLICATION OF THE NGS FOR THE ANALYSIS OF SEA EPIBIONT CLUSTERS ACCORDING TO CHANGES IN WATER TEMPERATURE Han Seung Lee1, Da- eun Nam1, Minkyu Choo1, Namyul Kim1, Hyunjung Joo1, Taegyu Kim1, Bongjin Jeikal1

1 Korea coast guard research center, Forensic science research team, Cheonan-si Chungcheongnam-do, Republic of Korea

The interest in forensic entomology on land has continued since 1850s, and in 1960s scientific research on the topic was performed focusing on animal cadavers. Forensic entomology only allows estimation of the time of death for persons who died of unnatural death on land. Its application to persons who have died on the sea or to evidence thrown into the sea is limited. This study focused on a method, similar to forensic entomology, for estimation of the time when evidence entered the sea water by using the specific characteristic of sea epibionts of remaining at the place of initial attachment for the rest of their lives. To observe the initial epibionts according to water temperature in the Busan area of the Republic of Korea, a division was made between the periods of low temperature and rising temperature, and it has been verified that there are differences between the species of sea epibionts in the bottom layer and surface layer. One of the NGS (Next Generation Sequencing) technique, Miseq was performed by sampling the DNA of marine organisms attached to identical size. Gene sequence of the marine organisms was analyzed using universal marker and the patterns for emergence of the main species of marine organisms according to water temperature were analyzed. An additional analysis of the epibiont species emerging in periods of high water temperature and periods of falling temperature will be performed and an observation focusing on the differences according to water depth and temperature is scheduled. The materials of this study can be utilized not only in the area of forensic science but also for research of local marine ecological environment, research on ecological changes caused by warming of the Korean peninsula or as distribution data on marine resources.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 105 P025 - ASSESSMENT OF INDIVIDUAL SHEDDER STATUS AND BACKGROUND DNA ON OBJECTS: DIRECT OR INDIRECT TRANSFER? Mariana Rolo1,2, Lisa Sampaio1, Filipa Balsa1, Ana Margarida Bento1, Nair Gouveia1, Armando Serra1, Pedro Brito1, Virgínia Lopes1, Marta São Bento1, Vanessa Bogas1, Patrícia Cunha1, Maria João Porto1, Maria José Carneiro de Sousa2

1 National Institute of Legal Medicine and Forensic Sciences- I.P., Forensic Genetics Service - Centre Branch, Coimbra, Portugal 2 University of Porto, Institute of Biomedical Sciences Abel Salazar, Porto, Portugal

Improvements in the sensitivity of DNA profiling scientific techniques allowed the analysis of limited amounts of DNA, often from multiple individuals, and the testing of not only body fluids, but also DNA deposited through contact on surfaces – touch DNA. The possibility of DNA transfer often raises the question of how the DNA was deposited: directly or by indirect transfer? Therefore, it is relevant to understand the factors that can influence these transfers. This investigation aimed to study (1) the direct transfer of touch DNA, by assessing the shedder status of an individual, and if it could be detected using normal procedures for casework samples, and (2) the background DNA present on some objects, utilized in an office environment, from volunteers. Ten volunteers were instructed to place their right thumb on a pre-cleaned glass plate, applying pressure and friction, and, on a different occasion, to hold a sterilized plastic tube. Swabs were also performed on objects that were regularly used for at least a month. All experiments were carried out in triplicate, samples were collected using a moistened swab and processed using commercial kits. Preliminary results show that for the direct transfer experiment, when DNA was detected, the majority of samples were single source profiles from the donor. However, non-self DNA was detected in some of the samples indicating secondary transfer. For the background DNA experiment, although the samples were from personal objects, almost all of them were mixtures. Once touch DNA evidence is currently accepted by the criminal justice community as a sample capable of providing an useful genetic profile, sharing data between laboratories and researchers is of importance for a better understanding on this subject.

106 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P026 - AUTOMATION OF HIGH VOLUME MPS MIXTURE INTERPRETATION USING CASESOLVER Øyvind Bleka1, Rebecca S. Just2, Jennifer Le2, Peter Gill1

1 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway 2 FBI Laboratory, DNA Support Unit, Quantico, USA

For very serious crimes, reporting scientists often have to contend with complex investigations, where literally hundreds of items are submitted by investigators for analysis per individual case. In order to efficiently expedite the challenge of comparing reference profiles to evidence profiles, many of which are mixtures of two or more individuals, we have developed an investigative open-source expert system ‘CaseSolver’ (www.euroformix/casesolver). The software provides an overview of contributors in common to multiple case stains (both single sources and mixtures) in a user-friendly graphical interface. In addition to handle Short Tandem Repeats (STRs) data, the software can also handle Massive Parallel Sequencing (MPS) data where sequence reads are utilized. We provide a demonstration using samples from a validation study analysed with the Il- lumina ForenSeq kit. By automating the process, CaseSolver removes the very time-consuming aspect of carrying out hundreds of comparisons with conventional probabilistic software, and gives improved quality by preventing manual errors. A comparison of results of using different levels of nomenclature was carried out using: raw strings, ordinary repeat units, and the longest uninterrupted stretch (LUS) nomenclature variant. We report on the advantages of enhanced discrimination offered by MPS when complex mixtures are analysed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 107 P027 - BACKGROUND DNA ON FLOORING: THE EFFECTS OF CLEANING Jack Reither1, Emma Gray1, Annalisa Durdle1, Xavier Conlan1, Roland van Oorschot2,3, Bianca Szkuta1,2

1 Deakin University, School of Life and Environmental Sciences, Geelong, Australia 2 Victoria Police Forensic Services Centre, Office of the Chief Forensic Scientist, Macleod, Australia 3 La Trobe University, School of Molecular Sciences, Bundoora, Australia

The collection and detection of ‘background’ DNA (BDNA) from items submitted for forensic DNA sampling is a common occurrence when sampling ‘targeted’ DNA left behind following an activity of interest. There are a number of factors that might affect the presence of BDNA on a surface such as the duration and history of prior use(s), shedder status of the user(s), substrate interactions, environmental conditions, and cleaning of the surface. Here, the impact of household cleaning methods on the presence of BDNA in five homes occupied by the same known individuals (all ≥18 years of age) for at least 3 months prior to sampling has been investigated. Each home had at least two occupants, and residents were not directly related to one another. Samples were collected from areas of high and low activity on floors within the kitchen, living room, bedrooms and bathrooms, and questionnaires provided information relating to the properties of the surfaces and the history of their use, including how, when and by whom they were last cleaned. Adjacent samples were taken pre- and post-cleaning from each activity area within rooms. Floors were cleaned by the participant according to their normal routine, with methods and products varying between rooms and homes. Overall, greater quantities of DNA were obtained from samples taken prior to cleaning compared to post-cleaning. Further, major contributors were more frequently assigned in profiles from samples obtained pre-cleaning compared to post-cleaning, although the number of contributors was generally greater in profiles from samples pre-cleaning. This study demonstrates the effects of cleaning methods on BDNA and highlights the need to consider cleaning when addressing activity related matters.

108 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P028 - BIRD OF PREY CE & MPS MULTIPLEXES: HIGH DISCRIMINATION FOR FORENSIC AND CONSERVATION APPLICATIONS Jordan Beasley1, Jon Wetton1, Celia May1

1 University of Leicester, Genetics and Genome Biology, Leicester, United Kingdom

Bird of prey or raptor persecution is one of the six priorities for the UK National Wildlife Crime Unit. Shooting and destruction of raptors account for the largest number of incidents reported to the UK Royal Society for the Protection of Birds, with 1,225 incidents between 2010 and 2015 [1]. Theft from the wild for falconry and illegal trade is also a serious problem [2] and can threaten natural populations as adult survival rate has a severe impact on population dynamics [3]. It is noteworthy that in 2010, Belgian Police seized over 700 birds of prey because of falsified CITES documents [4]. However, securing any prosecution for these crimes is often difficult due to a lack of evidence regarding identity or relatedness, e.g. comparing chicks stolen from a wild nest to alleged captive parents and siblings, or linking blood-stained clothing with the trapping or disposal of a specific bird. This project therefore aims to design and validate multiplex systems for a wide variety of raptors both for wildlife crime and population genetics purposes. Given the evolutionary distance, a separate test is being developed for eagles, hawks and kites compared with that for falcons. The assays will target species and individual identification as well as sexing markers (i.e. mtDNA barcodes, STRs & Z/W sex chromosome sequences) and they are being developed for use both on traditional CE and MPS platforms for maximum uptake by the scientific and forensic communities. 2. Wetton, J.H. & Parkin, D.T, 1997. Mol Ecol, 6:119–128. 3. Whitfield, D.P. et al., 2004. Biol Cons, 119:319–333. 4. http://www.unodc.org/unodc/en/data-and-analysis/wildlife.html

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 109 P029 - BODE BIOSAFE SWAB: PREVENTING CRIME SCENE “CULTURE” FROM DEGRADING DNA EVIDENCE Dan Watsula1, Robert Bever1, Sayed Mosavi1, Allie Flores1, Jangbir Sangha1, Manzar Ahmed1

1 Bode Technology, Products, Lorton, USA

A combination of new technologies introduced into the market and the increasing impact of the use of DNA to solve crimes has led to both increased reliance on DNA and more collections. One often overlooked area is the impact that biological materials, collected along with the samples at the crime scene, can have on the resulting DNA profile. Bacteria, fungi, and enzymes, such as DNases, can have a dramatic impact on the stability of a DNA sample possibly degrading it before it reaches an analyst’s bench for extraction and amplification. This presentation will describe the studies performed on mock crime scene samples to examine the specific effects of microbes and enzymes on the collected samples, the resulting DNA profiles, and introduce a point of collection preservative system; The Bode BioSafe Swab. Biological materials were deposited on various mock crime scene surfaces. After deposition, the samples were placed outside in the environment for a few days to simulate and stimulate normal bacteria, fungal, and enzymatic activity that can occur at a crime scene. The samples were collected using a standard cotton swab and placed in a swab box or using The Bode Bio- Safe Swab. Real time and accelerated aging experiments were performed on the samples by placing them in various temperature and humidity conditions. Significant degradation of biological evidence can occur in a very short period when the sample is not optimally collected and stored. The Bode BioSafe swab decreased sample degradation and increased the number of complete profiles obtained. The full results presented from this study will include MPS data on the different bacterial species collected alongside the genetic evidence, the quantification and degradation index values, and DNA profiling success rates.

110 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P030 - BONE SAMPLING CRITERIA FOR DNA GENOTYPING: MACROSCOPIC SAMPLE CATEGORIZATION AND STR TYPING RESULTS ASSOCIATION Andrea Natalia Rocha1, Micaela Longaray1, Carola Romanini1, Magdalena Romero1, Martina Rotondo1, Maria Laura Catelli1, Carlos Vullo1

1 Argentine Forensic Anthropology Team EAAF, Forensic Genetic Laboratory LGF-EAAF, Cordoba, Argentina

The Argentine Forensic Anthropology Team (EAAF) investigates human rights violations which took place in Argentina during the military government that ruled from 1976–1983 and cooper- ates with other countries as well. Because of this, the skeletal remains the EAAF processes may have been exposed to different moisture, temperature and pH conditions, as well as diverse soil types and the presence of microorganisms depending on their origin. There are other conditions that can turn the skeletal elements analysis even more complex, such as the disarticulation of bodies or severely burnt remains, among others. All mentioned above influence DNA quantity and quality. In the present study, we categorized the samples based on their macroscopic aspect, in order to investigate if there is a correlation between the aspect of the remains and the probability to obtain a genetic profile. Samples were classified into four major groups: score 1 was assigned to samples showing very good state of preservation, score 2 to those that were still well preserved but with slight superficial alterations. Score 3 and 4 involved regular and bad macroscopic appearance respectively. The data found clearly support the hypothesis that samples with a good score show a high concentration of well-preserved DNA, which leads to a greater reportable number of STR markers in comparison to those ranked in the worst categories. The purpose of this work is to provide a useful and practical categorization to perform sampling criteria that will lead to good genetic results for cases where there is the possibility of choosing the remains to analyse.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 111 P031 - BUSTING THE MYTHS: DNA TYPEABILITY AFTER 48 HOURS OF BOIL Eva Tikalova1, Jitka Votrubova1, Pavla Rihova2, Daniel Vanek1,3,4

1 Forensic DNA Service, DNA laboratory, Prague, Czech Republic 2 Czech Inveronmental Inspectorate, Cites, Prague, Czech Republic 3 Charles University in Prague, 2nd Faculty of Medicine, Prague, Czech Republic 4 Institute of Legal Medicine- Bulovka Hospital, DNA laboratory, Prague, Czech Republic

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades its use has become more widespread outside of Asia. One of the most trafficked products of TCM is the broth from Panthera tigris (PT). The traditional recipe for the preparation of Panthera tigris broth requires to boil the PT bones with removed bone marrow for 7 days and 7 nights. The final PT broths analyzed in our laboratory in the past did not yielded any results usable to perform the DNA based specie identification. The aim of our study was to monitor the quality and quantity of DNA in bone samples boiled for 48 hours. The sampling of Bos taurus bone disks and bouillon was done every hour for 2 days. The subsequent DNA analysis used multiple mtDNA targets (100–700 bp) to evaluate the quality and quantity of DNA extracted. DNA extracted from bone disks remained typeable after 48 hours of boil.

112 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P032 - CANINE MITOCHONDRIAL INVESTIGATION: A CALL FOR ALL DOGS Federica Giangasparo1, David Ballard1, Brian Catchpole2, Denise Syndercombe Court1

1 King’s College London, King’s Forensics, London, United Kingdom 2 Royal Veterinary College, Pathobiology and Population Sciences, London, United Kingdom

Our animal friends commonly share our living spaceare more and more frequently found in our housesOur animal friends are more and more frequently found in our houses making them one of the biggest DNA shedders in the home. In a crime scene, hairs specimens are often of animal derivation and they can be retrieved also from item of clothing or be left at a scene of crime in a secondary transfer event. Being able to understand from the hair whether the specimen is animal and if it is of canine origin and being able to discriminate determine the breed of the dog could provide important intelligence that will help the investigations. In the case of a hair sample being retrieved, often mitochondrial DNA analysis is the investigative method of choice. Recent research has demonstrated that the canine control region is not discriminative enough to infer the breed of origin, proposing the need for a full mitochondrial genome approach. The aim of my research is to build a full genome mitochondrial database to be able to evaluate the sequence variations and haplotype frequencies and with the aim of being able to infercanine breed.from the collected data whether breed of origin can be predicted for each individual dog. In order to gain confidence in this process, a many sufficient number of samples must be collected per breed to increase the statisticalsignificance of the results. A collection of 900 samples representing 87 breeds have been sampled through a collaboration with the aid- withof the Royal Veterinary College. Here I present the data from the first 200 samples analysed along with their cladistic distribution.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 113 P033 - CASEWORK EXPERIENCE USING mRNA ANALYSIS FOR HUMAN BODY FLUID AND TISSUE IDENTIFICATION Maximilian Neis1, Magdalena Bogus1, Gabriele Förster1, Kerstin Schöbel1, Cornelia Schmitt1, Markus A. Rothschild1, Peter Schneider1

1 Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany

The identification of human body fluids and tissues in criminal casework has been significantly improved by using a combined mRNA/DNA analysis. During the last couple of years, assays have been developed based on micro-RNA or mRNA for the specific detection of typical body fluids frequently observed e.g. in sexual assault cases. We have used an approach based on DNA/RNA co-extraction followed by standard STR typing for the DNA fraction, and by reverse transcription from total RNA, respectively. The resulting cDNA was subjected to end-point PCR using fluorochrome-labelled primers and detection by capillary electrophoresis of the following body fluid and tissue markers: blood, menstrual blood, vaginal secretion, semen, saliva, and nasal secretion, and including two sex-specific RNA markers, according to a modified protocol established by the Netherlands Forensic Institute (NFI; van der Berghe et al., Forensic Sci Int Genet. 2016; 20:119–129; Forensic Sci Int Genet. 2017; 26:70–76). Using a multiplex consisting of up to 19 PCR primer pairs, we have analyzed several sexual assault cases involving various scenarios of abuse with and without violence. In all cases, we were able to detect specific body fluid markers indicating the presence of saliva, vaginal secretion, blood or menstrual blood, depending on the circumstances. Our results were submitted as evidence in criminal and judicial proceedings demonstrating the usefulness of these methods.

114 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P034 - CHARACTERIZATION OF MITOCHONDRIAL AND NUCLEAR DNA IN SINGLE SHED HAIRS Michael Brandhagen1, Odile Loreille1, Jodi Irwin1

1 FBI Laboratory, DNA Support Unit, Quantico- VA, USA

Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, nuclear DNA short tandem repeat (STR) profiling is generally unsuccessful, and DNA testing of shed hair is instead performed by targeting the mitochondrial DNA control region. The high copy number of mitochondrial DNA relative to nuclear DNA routinely permits the recovery of mtDNA data in these cases. How- ever, mtDNA profiles do not offer the discriminatory power of nuclear DNA profiles. In order to better understand the total content and degradation state of DNA in single shed hairs, and assess the feasibility of recovering highly discriminatory nuclear DNA data from this common evidence type, high throughput shotgun sequencing was performed on both recently collected and aged (approximately 50 year-old) hair samples. The data reflect trends that have been demonstrated previously with other technologies, namely that mtDNA quantity and quality decrease along the length of the hair shaft. In addition, the shotgun data reveal that nuclear DNA is present in shed hair and surprisingly abundant relative to mitochondrial DNA, even in the most distal fragments. Here, we characterize both the nuclear and mitochondrial DNA content of shed hairs and discuss the implications of these data for forensic investigations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 115 P035 - CHARACTERIZATION OF NEW CHLOROPLAST POLYMORPHISMS TO DETERMINE BIOGEOGRAPHICAL ORIGIN AND CROP TYPE OF CANNABIS SATIVA SAMPLES Madeline G. Roman1, Michele Di Nunzio2, Rachel Houston1, Bobby LaRue1, Carme Barrot Feixat2, David Gangitano1

1 Sam Houston State University, Forensic Science, Huntsville, USA 2 University of Barcelona, Public Health, Barcelona, Spain

Marijuana (Cannabis sativa) is the most commonly used illicit drug in the United States. Despite its schedule I classification by the federal government, 33 states have legalized its use for me- dicinal or recreational purposes. This state-specific legalization has created a new problem for law enforcement: preventing and tracking the diversion of legally-obtained cannabis to states where it remains illegal. In addition, illegal trafficking of the drug at the border with Mexico remains an issue for law enforcement agencies. C. sativa crops can be broadly classified as marijuana (a drug containing the psychoactive chemical delta-9-tetrahydrocannabinol) or hemp (the non-drug form of the plant). Differentiation between crop types is important for forensic purposes. In addition, investigation of trafficking routes into and within the United States requires genetic association of samples from different cases/seizures, and knowing where the crop originated is extremely useful. This project seeks to exploit sequence variations in C. sativa chloroplast DNA (cpDNA) to allow genetic determination of biogeographic origin, discrimination between drug-type Cannabis and fiber-type Cannabis samples (marijuana vs. hemp), and association between cases for C. sativa samples. By comparing published cpDNA sequences, 58 polymorphisms have been identified, as well as seven hotspot regions. In this ongoing project, hemp and marijuana samples from four countries (United States, Canada, Mexico, and Chile) have been evaluated for informative polymorphisms in two cpDNA hotspot regions. Principal component analysis confirmed differences between the groups based on their crop type and biogeographic origin.

116 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P036 - CircRNA: A NOVEL BIOMARKER FOR FORENSIC AGE ESTIMATION? Junyan Wang1, Guangping Fu2, Qian Wang2, Bin Cong2, Shujin Li2

1 Hebei Key Laboratory of Forensic Medicine, Forensic Medicine- Hebei Medical University, shijiazhuang, China 2 Hebei Key Laboratory of Forensic Medicine, Department of Forensic Medicine- Hebei Medical University, Shijiazhuang, China

Molecular biomarkers can be useful for providing physical characteristics such as gender and age. Studies have shown that circular RNAs (circRNAs) upregulate globally during aging in multiple organisms. In this study, we collected peripheral whole blood samples from thirteen unrelated healthy volunteers (aged between 20 and 62 years) and analyzed the circRNAs expression profiles using deep RNA sequencing technology. 56,330 circRNAs were identified among these samples. Simple linear regression was performed using “lm” function of R package. Cor- relation coefficients (R) between age and circRNAs’ expression were calculated for each single circRNA site to identify the age-associated circRNAs (R<-0.8 or R>0.8) that will be further used to build age prediction model. We finally identified 20 circRNA candidates which show significant change during aging (12 downregulate and 8 upregulate). hsa_circ_0007356 was found to be most significantly correlated with age (R2=0.956, P=2.78×10-5). In light of this, we propose that circRNA could be used as a novel biomarker for individual age estimation. These 20 selected age-associated circRNAs will be further validated in 200 unrelated volunteers with ages of 20–70 years by RT-qPCR.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 117 P037 - COMPARATIVE ANALYSIS OF AUTOMATED DNA EXTRACTION PROCEDURES FOR USE IN FORENSIC CASE WORK Hye Jin Lee1, Ji-Young Kim1

1 Criminal Investigation Command- Ministry of National Defense, Division of DNA Analysis- Scientific Investigation Laboratory, Seoul, Republic of Korea

Advances in DNA profiling technology allow for the analysis of minute quantities of DNA. It is often possible to obtain successful short tandem repeat (STR) profiles from biological material transferred from an individual. Trace DNA analysis is a significant part of a forensic DNA testing laboratory’s workload. The successful recovery of trace DNA is highly variable. It seems to be dependent on a wide range of factors, from the characteristics of the donor, item types and environment, to DNA extraction methods. Adopting optimal DNA extraction procedure for trace DNA have significant influence on successful STR analysis. To introduce new DNA analysis method to forensic DNA testing laboratories, reliability of the procedure needs to be demonstrated by carrying out internal validation or verification process. When the process is conducted properly, DNA testing results obtained by using the analysis method can be a useful investigative tool for the law enforcement community. In this study, in order to introduce an automated DNA extraction method for evidence items commonly encountered in forensic casework including buccal swab, blood stain, cigarette butt, and trace DNA, a comparative analysis for three DNA extraction protocols using Maxwell RCS48 system was performed. Validations of these DNA extraction protocols include all of the studies required by the validation guideline. The optimal protocol for trace DNA was validated for reproducibility, robustness, and performance such as yield, purity, and concentration.

118 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P038 - COMPARISON OF DNA YIELD AFTER LONG-TERM STORAGE OF SECOND WORLD WAR BONE SAMPLES Irena Zupanič Pajnič1, Sara Grdina1, Eva Lina Friš1, Jože Balažic1, Eva Podovšovnik2

1 University of Ljubljana, Medical Faculty- Institute of Forensic Medicine, Ljubljana, Slovenia 2 University of Primorska, Turistica - Faculty of Tourism Studies, Portorož, Slovenia

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enable successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing person from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future if necessary. After molecular genetic analyses of Slovenian WWII victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples effects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analysed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Bone powder was obtained from 54 WWII femurs and half of gram decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Up to 100 ng DNA/g of bone powder was obtained from femurs in 2009 and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 119 P039 - COMPARISON OF FOUR SWAB TYPES FOR STAIN COLLECTION Jonathan Währer1, Agata Knap1, Sarah Kron1, Ilona Blank1, Iris Schulz1

1 Institute of Legal Medicine, Forensic Genetics, Basel, Switzerland

The collection and recovery performance of a swab is critical to the total amount of DNA available for the subsequent processes and ultimately determines the STR profiling success rate. Therefore, we have started a collaboration study with three Swiss police departments to determine the optimal swab for our laboratory processes. We are examining 1) possible quality differences in swab types, 2) possible correlations between swab type and stain type, 3) possible differences in DNA conservation due to swab storage, and 4) possible sampling efficiencies of DNA collectors. The focus of our study is particularly on the investigation of touch DNA on different swab types. For the overall picture, however, we have also included body fluids expecting possible differences in DNA quantification, but not in the typing success rates. For the collaborative study, our laboratory prepared traces with homogeneous amounts of DNA on artificial evidence frequently used in burglary offences. DNA was collected from the prepared stains (n = 624) according to our instructions by various police workers and our staff using four different swab types (Copan 4N6FLOQSwab Genetics, Copan 4N6FLOQSwab CrimeScene, Pri- onics Evidence Collection Kit, Prionics ForensiX SafeDry). DNA from the swabs of all collectors (police and our staff) were then extracted (Promega Maxwell®), quantified and amplified with the commercial kits Plexor® HY and PowerPlex® ESI 17 Pro System (Promega). We will present our unexpected pre-test result of a weak performing swab and give an overview of the swab type related differences in DNA quantity and quality and discuss the potential effects of the stain types and skills of the DNA collectors.

120 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P040 - CREATING A METAGENOMIC ‘DNA MAP’ OF THE LONDON UNDERGROUND TRANSIT SYSTEM Gabriella Mason-Buck1, Pawel Labaj2, David Ballard1, Rebecca Smith1, Dedan Githae2, Wojciech Branicki2, Denise Syndercombe Court1

1 King’s Forensics, King’s College London, London, United Kingdom 2 Małopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland

It is estimated that 5 million passengers travel on the London underground network daily. It is the oldest and one of the largest underground network systems in the world; 270 stations across 11 lines, spanning 402 km. Previous studies have implied that locations have unique metagenomic fingerprints but not much is currently known about the metagenomic ecosystem of underground transit systems. The Mason Lab at Weill Cornell University formed The Metagenomics and Metadesign of Sub- ways and Urban Biomes (MetaSUB) International Consortium to investigate this further. This consortium unites over one-hundred scientists from more than 50 cities across all continents with the goal of examining mass-transit system microbiomes to construct a world-wide ‘DNA map’. To achieve this, we carry out a global City Sampling Day (gCSD) annually on the 21st of June. On this day, each city within the consortium deploys a team of scientists and citizen scientists to swab areas on the transit systems. In London, a bench on the platform, a handrail or eleva- tor button and a ticket machine were swabbed at each station. The swabs were then sent to the USA for sequencing. The sequencing data will enable the creation of geospatial metagenomic and forensic genetic maps. Promising results have been obtained from the MetaSUB data both by ongoing analysis within the consortium as well as by external scientists taking part in the Metagenomic Foren- sics Challenge at the Critical Assessment of Massive Data Analysis (CAMDA) conference. Within the consortium we are also developing ways to visualise this complex data (MetaGenScope) as well as constructing guidelines on how to communicate the results to the public. Data from the London MetaSUB gCSD 2017 will be presented.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 121 P041 - CUTANEOUS miRNAS EXPRESSION IN HUMAN HANGING INJURIES Matteo Fabbri1, Paolo Frisoni1, Matteo Marti1, Letizia Alfieri1, Enrica Calabrese1, Chiara Marini1, Raffaella Marino1, Rosa Maria Gaudio1, Margherita Neri1

1 University of Ferrara, Department of Morphology- Surgery and Experimental Medicine - Institute of Legal Medicine, Ferrara, Italy

The determination of dermal injuries age is crucial in forensic caseworks. Despite the importance of the topic, there currently exists no molecular methods to estimate dermal injury age. Our preliminary work evaluated one of the most frequent injury detect in our casework records and represented by hanging injury. Samples were represented by 40 hanging injuries and 10 control samples. For all the selected subjects skin diseases were excluded. All skin biopsies were treated using 500mL of RNAlater tissues stabilizer (QIAGEN) in order to avoid miRNAs degradation. MiRNAs were extracted using miRNeasy Mini kit (QIAGEN) following the manufacturer’s protocol. MiRNAs were eluted in 14 mL of RNase-free water and stored at -20 °C prior to reverse transcription. Reverse transcription was carried out using the miScript II RT kit (QIAGEN). cDNAs generated with the miScript II RT Kit were used as a template for a real-time PCR accomplished by the miScript miRNA PCR Array - Human Cell Differentiation & Development (QIAGEN) on an 7500 Real-time PCR platform (Applied Biosystems). The selected array format was enable

to detect 84 specific miRNAs. Data analysis were carried out applying the ΔΔCt method and using the data analysis software tool provided by the manufacturer (QIAGEN). Hanging injury samples showed an overexpression (p value < 0.05) of miR125a-5p e miR125b-5p, miR128–5p, miR214–3p, miR133b, miR206, miR122–5p, miR103a-3p, miR15–5p, miR150–5p and miR92a-5p. These markers are involved in the regulation process occuring in the inflammation phase in which inflammatory citokynes are inhibited. The expression patterns of the detected markers suggest they have potential to predict injury age (hours), especially during the initial stages of injury healing.

122 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P042 - DEAD MIGRANTS IN THE MEDITERRANEAN: GENETIC ANALYSIS OF BONE SAMPLES EXPOSED TO SEAWATER Emilie Bertolini1,2, Pierangela Grignani2, Barbara Bertoglio1,2, Cristina Cattaneo1, Debora Mazzarelli1, Agata Iadicicco3, Alessandro Bosetti4, Paolo Fattorini5, Silvano Presciuttini6, Giorgio Marrubini7, Carlo Previderè2

1 Università di Milano, LABANOF- Dip. Scienze Biomediche per la Salute, Milan, Italy 2 Università di Pavia, Dip. Sanità Pubblica- Medicina Sperimentale e Forense, Pavia, Italy 3 Ufficio del Commissario Straordinario per le Persone Scomparse, Ministero dell’Interno- Roma, Rome, Italy 4 Promega Italia, Milano, Milan, Italy 5 Università di Trieste, Dip. Clinico di Scienze Mediche- Chirurgiche e della Salute, Trieste, Italy 6 Università di Pisa, Dip. Ricerca Traslazionale e Nuove Tecnologie in Medicina e Chirurgia, Pisa, Italy 7 Università di Pavia, Dip. Scienze del Farmaco, Pavia, Italy

On April 18th 2015 a medium size fishing boat sank, just off Lybian waters, drowning hundreds of migrants packed on board. The victims were believed to be trapped in the cargo, 400 meters below sea level. The Italian government created a task force for the recovery and identification of the victims. In July 2016, the boat was recovered and 528 bodies (skeletal remains) were retrieved along with many commingled remains belonging to about 200 individuals. A selected sample composed of 80 victims was chosen on the basis of the finding of any document recovered by a clothing inspection that suggested a possible identity of the victim. DNA was extracted from femoral and tibial diaphyses. Three different DNA extraction methods were used (Auto- mated extraction with DNA IQ casework PRO kit for Maxwell 16, Promega; Prepfiler BTA forensic DNA extraction kit, Thermofisher; Qiamp DNA Blood Maxi kit, Qiagen). DNA quantification was performed using Quantifiler Duo kit (Thermofisher) and provided DNA amounts below the limit of quantification (< 23 pg/µl) in more than 80 % of the samples. In order to get 21 autosomal STR profiles, we planned to amplify the DNA extracts using three kits with different multiplex combinations (PowerPlex ESX 17 Fast System, PowerPlex ESI 17 Fast System, AmpFl STR Iden- tifiler Plus). This allowed to cross-check results from poor-quality samples. In conclusion, about 80 % of the genetic profiles were composed by at least 16 STRs and about 25 % were complete 21 STRs profiles. Statistical analyses were performed in order to identify which factor between time of the recovery of the bodies, position in the boat, and state of preservation of the bones, may have affected the quality of the genetic profiles.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 123 P043 - DETERMINATION OF AN EFFECTIVE INTERNAL REFERENCE GENE FOR THE QUANTIFICATION OF mRNA FOR BODY FLUIDS IDENTIFICATION Yixia Zhao1, Anquan Ji1, Sheng Hu1, Caixia Li1, Le Wang1, Qifan Sun1

1 Institute of Forensic Science-Ministry of Public Security, MPS’s Key Laboratory of Forensic Genetics- National Engineering Laboratory for Crime Scene Evidence Investigation and Examination-, Beijing, China

mRNA profiling has been under scrutiny over the past couple of years for its potential ability to identify a variety of body fluids in forensic samples. Constitutively expressed housekeeping genes would be used as a reference for RNA quality control and normalization, for assessing the expression of target mRNA from various biological samples. Given the increased sensitivity and reproducibility, the requirements for a proper internal control gene for normalization have become very important. In the current study, we selected three internal genes based on previous study, including GAPDH, beta-actin, S15, and aimed to identify the most stably expressed control gene in peripheral blood, menstrual blood, vaginal secretions, semen. Total RNA was extracted from four body fluids. RT-PCR products were separated and detected by capillary electrophoresis. The results showed that GAPDH had the highest fluorescence intensity in peripheral blood and menstrual blood, followed by peripheral blood, and was weak in vaginal secretions. The fluorescence intensity of β-actin showed the same trend as GAPDH, but the overall fluorescence intensity was weaker. The S15 just detected in the peripheral blood, menstrual blood and semen, the fluorescence intensity is highest in the menstrual blood. The results indicated that the ubiquitously expressed internal reference genes GAPDH and β-actin were detected in all four biological stains, among which GAPDH was the most highly expressed and had the best specificity. S15 was widely expressed in peripheral blood, menstrual blood and semen, but the overall expression was worse than GAPDH and β-actin. Therefore, GAPDH and β-actin can be preferentially selected as internal reference genes mentioned above.

124 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P044 - DETERMINATION OF DNA YIELD RATES IN SIX DIFFERENT SKELETAL ELEMENTS IN ANCIENT BONES Živa Miriam Geršak1, Irena Zupanič Pajnič2, Matija Črešnar3, Jože Balažic2

1 University Medical Centre Ljubljana, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 University of Ljubljana, Medical Faculty- Institute of Forensic Medicine, Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Arts- Department of Archaeology, Ljubljana, Slovenia

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femurs, teeth and temporal bones. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from eight different skeletons (six from 18th century and two from 3rd century) were obtained from five archaeological sites in Slovenia. In each skeleton, six different skeletal elements were sampled (temporal bone, molar, femur, metacarpal and metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half gram of powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analysed. The highest yields were detected in temporal bones and the lowest in femurs. The success rate of STR typing was evaluated according to the number of successfully typed loci and strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons, full consensus genetic profiles were determined from skeletal elements analysed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 125 P045 - DEVELOPMENT OF A BODY FLUID-SPECIFIC mRNA MULTIPLEX PANEL: A NEW APPROACH BASED ON WTA FOR GENOTYPING FORENSIC TRACE SAMPLES Qiuyue Wang1, Zehua Gao1, Ting Fang1, Yuhan Hu1, Yiwen Yang1, Yuguo Huang1, Yueyan Cao1, Yijun Zhou1, Qiang Zhu1, Yufang Wang1, Ji Zhang1

1 West China School of Basic Medical Sciences and Forensic Medicine- Sichuan University, Department of forensic genetics, Chengdu, China

In forensic science, body fluids are common materials providing important clues to forensic caseworks. Several kinds of RNA genetic markers have been established to identify the source of body fluids, such as mRNA and microRNA. However, because of degradation and other environmental factors, the amount of body fluids discovered and obtained from the crime scenes is often low and cannot be detected by current testing methods. In this study, we constructed a multiplex amplification system for seven kinds of body fluids. Moreover, based on the whole transcriptome amplification (WTA), our study improved the detection sensitivity for micro body fluids through the reverse transcription of RNA and the pre-amplification of cDNA.

126 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P046 - DEVELOPMENT OF A MULTIPLEX REAL-TIME PCR SURVEILLANCE ASSAY FOR MONITORING THE HEALTH STATUS OF ECUADORIAN AMPHIBIANS AT RISK OF EXTINCTION German Burgos Figueroa1, David A. Narváez-Narváez2,3, Claire Muslin4, Luis Fabián Naranjo Núñez4, Byron Freire-Paspuel5, Andrés Merino-Viteri6, Alexander Genoy-Puerto4

1 Escuela de Medicina- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 Farmacia y Tecnología Farmacéutica y Fisicoquímica- Facultad de Farmacia y Ciencias de la Alimentación, Universitat de Barcelona, Barcelona, Spain 3 Grupo de Transporte y Vehiculización de Fármacos- Unidad de Tecnología Farmacéutica, Universitat de Barcelona, Barcelona, Spain 4 Escuela de Medicina Veterinaria- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 5 Laboratorios de Investigación, Universidad de Las Américas UDLA, Quito, Ecuador 6 Museo de Zoología QCAZ- Escuela de Ciencias Biológicas, Pontificia Universidad Católica del Ecuador, Quito, Ecuador

Ecuador is a tropical country with one of the highest indexes of biodiversity and endemism of amphibians in the world. Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of a TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bd, Bsal and ranavirus by using specific primers and fluorescent probes. Standard curves, with a high linear correlation (r2 > 0.995), were generated from a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens and were used as a standard curve templates. The assay was tested against different samples from skin, liver, kidney, spleen and lung from six different amphibian species, where the multiplex real-time PCR assay showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identify amphibian’s pathogens, represents a valuable tool for the early detection of these pathogens and infection monitoring for future epidemiological surveillance of amphibian species at risk of extinction. Moreover, it can be used to estimate the prevalence of this infections and co-infections of these pathogens.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 127 P047 - DEVELOPMENT OF A MULTIPLEX RT-PCR ASSAY AND ITS STATISTICAL EVALUATION FOR THE FORENSIC IDENTIFICATION OF VAGINAL FLUID Tomoko Akutsu1, Yokota Isao2, Watanabe Ken1, Sakurada Koichi3

1 National Research Institute of Police Science, First Department of Forensic Science, Kashiwa, Japan 2 Graduate School of Medicine- Hokkaido University, Department of Biostatistics, Sapporo, Japan 3 Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Department of Forensic Dentistry, Bunkyo-ku, Japan

The identification of vaginal fluid from casework samples of sexual assaults provides important probative evidence of vaginal intercourse. However, the specificity and detectability of previously reported markers or procedures seem to be insufficient for discriminating vaginal fluids from other body fluids. The aim of this study was to establish a more specific procedure for identifying vaginal fluid for forensic purposes. A multiplex RT-PCR assay was developed for simultaneous detection of vaginal fluid markers (ESR1, SERPINB13, KLK13, CYP2B7P1, MUC4), which were evaluated by quantitative RT-PCR assay. Each amplicon was separated and quantified automatically by chip electrophoresis. Detectability and cross-reactivity of the developed multiplex procedure were assessed using various forensically relevant body fluids. Then, a cutoff value for the positive detection of vaginal fluids was set for each marker to maximize the sum of sensitivity and specificity. The ability of the multiplex RT-PCR assay to distinguish between vaginal and other body fluids was evaluated statistically using a likelihood ratio (LR) that was determined using a Bayesian estimation approach that considered the infrequency of detection. A sufficiently high LR (LR=4.3×109 (95 % credible interval: 4.0×108 − 2.9×1012)) was obtained when all five markers showed positive results. The developed procedure was validated using vaginal fluid samples with various conditions. Relatively high LRs were found in aged vaginal fluid stains, although each amplicon peak became low. In conclusion, the multiplex RT-PCR-based procedure followed by the statistical evaluation using LR may prove to be a powerful tool for the objective identification of vaginal fluids.

128 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P048 - DEVELOPMENT OF AN INNOVATIVE APPROACH TO HUMAN DNA QUANTIFICATION ANALYSIS Priti Sabadra1, Jennifer Elliot1, Allison Holt1, Angela Lackey1, Scott Schroeder1

1 Thermo Fisher Scientific, Human Identification, S. San Francisco, USA

Quantifiler™ Trio human DNA quantification kits are more sensitive, resistant to inhibitors, include a more robust DNA standard, and provide sample quality and quantity information. The result of these changes is that the laboratory can more accurately predict downstream STR results including deciding whether to continue with STR analysis, which STR kit to use, and how much DNA to add to the STR reaction. Although improved and optimized, accurate quantification results still depend heavily on the standard curve generation and metrics. To further increase efficiency and decrease variability a flexible Virtual Standard Curve (VSC) feature was developed within the HID Real-Time PCR Analysis Software v1.3. VSC functionality allows the user to input standard curve quality metrics for each quantification target and analyze data with the user-defined values. This feature reduces quantification variation by negating the variation introduced during the creation of the standard curve dilution series. VSC increases productivity by allowing for the addition of more samples to each plate and saves analyst time. The use of optimized VSC protocols demonstrated how to decrease quantification variability while increasing efficiency. This study demonstrates a VSC can be utilized in a forensic DNA laboratory to improve accuracy and increase efficiency while not impacting STR results.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 129 P049 - DEVELOPMENT OF HUMAN OR ANIMAL IDENTIFICATION KIT Min-Hee Kim1, Gyeong Hyeon Kim1, Sehee Oh2, Ki Min Seong1

1 National Forensic Service, Forensic DNA division, Wonju-si, Republic of Korea 2 Busan institute- National Foresic Service, DNA analysis section, Yangsan-si, Republic of Korea

In this study, we aimed to confirm the presence of human or animal DNA from DNA isolated for human and animal identification kit development by multiplex real time PCR method. Therefore, the conventional method requires many experimental stages and labor, this study tries to make this easier. The basic idea is to use two mitochondrial DNA markers and two nuclear DNA markers to identify human or animals. For this reason, human leukocyte antigen gene as a human nuclear DNA confirmation marker and hypervariable region gene are used for mitochondrial DNA confirmation, and with animal DNA confirmation marker, β-actin for nuclear DNA confirmation and mitochondrial confirmation cytochrome B gene each was used to develop a test method. In this study, each primer set can be used to confirm individual human and animal DNA, and it is planned to explore the development of multiplex real time PCR and identification of animals and individual species by differences in Ct value through deepening studies. Through these studies, it is hoped that a method to more easily identify the source of DNA from isolated DNA will be useful for criminal investigations.

130 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P050 - DEVELOPMENT OF MULTIPLEX PCR SYSTEM FOR METABARCODING OF DIVERSE PLANT MIXTURE Hyehyun Oh1, Sungmin Kim1, Jee Young Park2, Hyun-Seung Park2, Hyeonah Shim2, Tae-Jin Yang2

1 Supreme Prosecutors’ Office, Department of Forensic Science Investigation - Division of Forensic Genetics and Chemistry, Seoul, Republic of Korea 2 Seoul National University - College of Agriculture and Life Sciences, Department of Plant Science - Plant Genomics and Breeding Institute and Research Institute of Agriculture and Life Sciences, Seoul, Republic of Korea

DNA barcoding has been widely applied for the authentication and detection of various raw materials. However, most of the forensic materials are in the form of mixtures in small amounts, requiring an advanced technique such as NGS based metabarcoding. Unlike animal or fungi, single barcoding regions for the identification of plants, such as matK or rbcL, do not show enough sequence variation covering the plant species diversity. In this study, we designed a multiplex PCR primer set in order to establish an efficient metabarcoding method capable of identifying components in various kinds of unspecified mixtures. For plants, we discovered several candidate barcoding regions from the chloroplast genome, which amplify all the diverse range of plants but showed enough internal sequence diversity. We also assessed their capability of being amplified in most plant species by evaluating PCR efficiency, species discrimination resolution, and amplification ability via in silico PCR. Multiplex NGS analysis was conducted for a diverse mixture of animal, plant, and fungi using six primers including four chloroplast primers for plants, 18s rDNA primer for animals and ITS primer for fungi. The amplicons of each primer were sequenced using the Illumina Miseq platform, and quantitative analysis was conducted by mapping the reads to the reference sequences. Our analysis will contribute to establishing a high throughput metabarcoding approach for qualification and quantification of original plant species in forensic materials as well as herbal products.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 131 P051 - DIFFERENT SKELETAL ELEMENTS AS A SOURCE OF DNA FOR GENETIC IDENTIFICATION OF SECOND WORLD WAR VICTIMS Marcel Obal1, Irena Zupanič Pajnič1, Barbara Gornjak Pogorelc1, Tomaž Zupanc1, Jože Balažic1

1 University of Ljubljana, Medical Faculty- Institute of Forensic Medicine, Ljubljana, Slovenia

To this day process of identification of missing persons from skeletonized human remains with help of forensic genetics proves to be complex and challenging. The success rate of genetic identification in bones strongly depends on a combination of various factors, most importantly environmental factors and post-mortem interval. Furthermore, there are individual-specific factors that affect DNA preservation, such as race, gender, age and type of skeletal elements. The goal of our study was to optimize sampling process through determining which skeletal elements are superior in their preservation of DNA in 70-year-old skeletons belonging to victims of Second World War. We sampled different types of bones and teeth from three such skeletons found in Slovenian hidden mass grave Huda jama, 56 elements from each respective skeleton, together 168 elements. With the help of parameters, such as quantity of DNA, degradation rate and typing success, we tried to find the best types of elements to identify the victims. Prior to powdering bones and teeth, we removed contaminants. We decalcified 0.5 g bone and tooth powder followed by extraction and purification of DNA using Biorobot EZ1 (Qiagen). Quantifica- tion of obtained nuclear DNA was carried out using PowerQuant kit (Promega) and autosomal STR typing using ESSplex SE QS kit (Qiagen). Best parameters to assess skeletal elements that are superior in their DNA preservation were quantity of DNA and number of successfully typed STR loci. Metacarpal and metatarsal bones proved to be the best, followed by intermediate cunei- form, first distal foot phalanx, talus, petrous bone and tibia. We also created elimination database for persons involved in exhumation, anthropological and genetic analyses and exclude potential contamination.

132 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P052 - DIFFERENTIATION OF BLOOD STAINS ORIGINATING FROM IDENTICAL TWINS BASED ON XENOBIOTIC ANALYSIS Jakub Czarny1, Łukasz Kaczorowski1, Bartosz Hornik1, Monika Antczak1, Magdalena Chrostowska2, Joanna Idkowiak1, Michał Raczkowski2, Natalia Galant2, Renata Herman2, Aleksandra Górka1, Jadwiga Musiał2, Jolanta Powierska-Czarny1

1 Institut of Forensic Genetics, Department of Forensic Genetics, Bydgoszcz, Poland 2 Institut of Forensic Genetics, Department of Forensic Toxicology, Bydgoszcz, Poland

The fact that identical twins possess identical genetic material is one of the dogmas of genetics. Although the results of metagenomic analyzes indicate slight differences in the structure of twins genomes, they have not been used yet in routine forensic analysis. We present a case study of a suicide or the murder and suicide of two male identical twins. In the case, a knife was secured with revealed blood spots on the blade and hilt and a genetic profile corresponding to the genetic profile of twins, and blood spots secured on clothing. Ex- traction of xenobiotics from the blood and blood spots was carried out using the liquid/liquid method. The study was carried out in the range of 521 psychoactive compounds, drugs and their residues using LC-MS/MS. The analysis was conducted by following two MRM pairs on a Sciex 5500 Qtrap spectrometer coupled with an Exiono liquid chromatograph on a Kinetex C18 column using a methanol/water phase system. Amphetamine was found in the blood of one of the men at a concentration of 386.5 ng/ml while THC and THCCOOH were found in the blood of the second at concentrations of 7.2 ng/ml and 85.1 ng/ml, respectively The presence of amphetamine was found on the blade of the knife, while THC and THCCOOH were found on the knife handle. Amphetamine or THC and THCCOOH or amphetamine and THC and THCCOOH were found in clothing stains. The results of the study show that the use of sensitive xenobiotics in blood spots may in some cases allow for the differentiation of blood traces from identical twins.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 133 P053 - DISCRIMINATION OF THE POISONOUS UROBOTRYA SIAMENSIS FROM THE GREEN-LEAF VEGETABLE ‘PAK-WAN’ Wanasphon Bunakkharasawat1, Laksika Panok2, Nathinee Panvisavas3

1 Faculty of Science, Mahidol University, Forensic Science Unit, Bangkok, Thailand 2 Faculty of Science, Mahidol University, Department of Plant Science, Bangkok, Thailand 3 Faculty of Science, Mahidol University, Forensic Science Unit / Department of Plant Science, Bangkok, Thailand

“Pak-wan” is a popular local edible green-leaf vegetable in South-east Asia. There are 2 species of Pakwan, i.e., Melientha sauvis PIERRE (MES) and Sauropus androgynous (SA). Villagers confuse the appearance of their edible young leaf and shoot with those of the poisonous plant, Uro- botrya siamensis Hiepko, and mistakenly picked. Ingestion of a meal-portion of this poisonous plant can lead to death within few hours. To confirm the cause of death, there is a need to identify this poisonous plant remains. However, it is difficult to identify the plant when lacking informative plant morphological characteristics. This study was then aimed to identify potential DNA markers of these 3 species for DNA-based identification. In this study, a total 203 samples from 3 plant species were collected from different parts of Thailand. Plant morphological characteristics were compared, especially, leave shape and apex, flower and fruit. DNA sequence analysis of 7 DNA markers showed sequence polymorphisms (SNPs and INDELs) in 5 potential DNA markers that can discriminate the 3 species, i.e. ITS, rbcL, trnT-L-F, trnH-psbA, and At103. In addition, 2 DNA markers; i.e., trnH-psbA and at103, revealed sequence length polymorphisms between the 3 species. TrnH-psbA fragment length of US, MES and SA were 300, 500 and 400 bases. At103 fragment length of US and SA were 400 and 600 bases. At103 was unsuccess- fully amplified from MES. For matK and sqd1 showed 80–100 % and 82–94 % sequence similarity which were insufficient DNA polymorphisms for identification. Further DNA test for the poisonous plant will be developed in the near future. The DNA test would benefit to both forensic and food safety issues.

134 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P054 - DNA EXTRACTED FROM SECOND CERVICAL VERTEBRA IS PREFERENTIAL FOR STR TYPING OF OLD HUMAN REMAINS Victor Kunin1, Elena Ioganson2, Olga Kravtsova1

1 Kazan Federal University, Dept. of Biochemistry, Biotechnology and Pharmacology, Kazan, Russian Federation 2 Bureau of forensic examination of the Republic of Tatarstan, Dept. of Forensic Biology, Kazan, Russian Federation

Introduction: DNA typing of old human remains can be limited by the stage of DNA extraction. Despite many extraction methods are applied for degraded samples, the question of preferential source of DNA for STR analysis is still open. Aim of the study: Herein we compared efficiency of autosomal STR analysis using DNA samples extracted from World War II victim’s burials. Materials and methods: DNA extraction was performed from 3 individual burials from tubular bones (femur, tibia, clavicle), flat bones (skull bones), several vertebrae (C2, C7, Th5, Th7, L3, L5, S1, S3) and coccyx by PrepFiler kit (Applied Biosystems, USA). DNA concentration measurement and autosomal STR typing was performed by Quantifiler Human DNA Quantification Kit and AmpFlSTR® Identifiler™ Plus from the same supplier. Results: Complete profile of autosomal STR loci was obtained in DNA samples extracted from specific structure of the 2nd cervical vertebra – denticular (or dental) process – special morphological structure of Atlanto-axial articulation – compared to partial data from the other DNA samples. To find out their particularities due to DNA preservation we’ve attempted its histological analysis. We’ve observed distinguishable osteons within Gaversov’s channels and the presence of lamellar structures in the bone beams which can serve as natural reservoirs for DNA storage. Furthermore, we analyzed histological structure of the same region from modern bone sample (after fire) and we’ve got the same picture – osteocytes are detected, which are evenly located inside the bone beams. Conclusion: We suppose that special structure of dental process creates optimal conditions for DNA preservation and this type of specimen can be optimal DNA source for further DNA profiling.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 135 P055 - DNA PROFILING SUCCESS RATES OF COMMONLY SUBMITTED CRIME SCENE ITEMS Hang Yee Wong1, Jiayu Tan1, Zi Gui Lim1, Rachel Yan Foong Kwok1, Wilson Wen Xiang Lim1, Shilen Ng1, Christopher Kiu Choong Syn1

1 Health Science Authority, Biology Division- DNA Profiling Laboratory, Singapore, Singapore

Our laboratory receives a myriad of exhibits from the law enforcement agencies annually, of which about one-third are trace articles which do not require serological testing for blood and semen. It should be recognized that not every exhibit will yield a reportable DNA profile. Various studies have reported on the success rate for different exhibits, but the list of exhibits studied are not fully applicable in Singapore’s context due to the difference in crime demographics. For example, paper with threatening messages, locks and chains are often encountered in harassment and unauthorized money lending cases, as well as cling film, aluminium foil and drug smoking apparatus which are commonly submitted from the drug crimes, but data on their DNA profiling success rates is limited. This pilot study aimed to examine the DNA profiling success of these exhibits which are unique to our local scene, as well as corroborate the results of other exhibits which have been previously studied. Data from 600 cases comprising over 1,100 exhibits for touch DNA analysis were collat- ed and mined for details such as types of article and types of DNA profile obtained. Data analytics was performed to identify the DNA profiling success rate of commonly submitted articles and also to examine the correlation between the type of articles submitted to the DNA profiling success rate of the different law enforcement agencies. It was observed that the types of exhibit submitted had an impact on the DNA profiling success rate of the respective law enforcement agencies. Results of this study can guide law enforcement agencies in identification and prioriti- zation of exhibits for DNA analysis, leading to higher success rate in DNA profiling and allowing for more effective utilization of resources.

136 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P056 - DNA TRANSFER PROBABILITIES REFLECTING DIFFERENT CASEWORK-RELEVANT SCENARIOS Frederike Tebbe1, Iris Buckel2, Eva Schultheiss2, Regine Banemann2, Ingo Bastisch2

1 Goethe-Universität, Molekulare Biowissenschaften, Frankfurt, Germany 2 Bundeskriminalamt, DNA-Analytik, Wiesbaden, Germany

DNA transfer has been a subject of forensic research for several years. The eventuality of indirect transfer – finding DNA evidence of an uninvolved, innocent person on touched items associated with a criminal case – has been an issue in court since very low amounts of DNA can successfully be analyzed.To improve the understanding of DNA transfer mechanisms many research teams performed various studies upon this complex topic. In this study we focused on primary and secondary transfer scenarios that are similar to real cases. One series of experiments evaluated the time dependence of secondary transfer after handshaking. A decrease of secondary transfer was observed already after short intervals. A second set of experiments assessed the secondary transfer through contact with worn scarfs and cardigans and used tissues. Tissues were shown to be good potential DNA vectors, whereas scarfs as well as cardigans were determined to be rather poor DNA vectors. A third experimental scenario targeted the possibility of depositing cells of a previous contact on zipper-bags and adhesive tapes that are often used as drug packaging. Here, primary transfer was observed almost consistently, however indirect transfer could not be observed in this scenario. These preliminary experiments provided general trends useful for the design of further studies with a greater set of test subjects and a higher number of samples to achieve significant results.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 137 P057 - EFFECT OF THE ACTIVITY IN SECONDARY TRANSFER OF DNA PROFILES Celia Jimenez1, Carlos Baeza-Richer1,2, Cláudia Gomes1,2, Sara Palomo-Díez1,2, Eduardo Arroyo‑Pardo1,2, Ana María López-Parra1,2

1 Medicine School Complutense University of Madrid UCM, Legal Medicine Psychiatry and Pathology Department, Madrid, Spain 2 Hospital Clinico San Carlos IdISSC, Instituto de Investigación Sanitaria, Madrid, Spain

Secondary transfer occurs when DNA is transferred from one object or person to another, via an intermediate object/person. Currently some lawyers are using the existence of this type of transfer as an argument to invalidate genetic analysis. In a recent case in Spain, the existence of secondary transfer was alleged from part of the defense to justify the presence of DNA on a specific towel, found in the crime scene. In this case, the towel with the defendant’s DNA was found in the house where the bodies of a father and his daughter appeared. The transference would have been made through the body of the wife since the accused had repeated sexual relations with her. In the present work, an attempt has been made to confirm the presence of a person’s DNA in an object, via an intermediate person,between individuals who are very often in contact, such as couples. It is intended to assess if, in those cases, it is possible to obtain a genetic profile after performing a current activity such as washing their hands. Two experiments were performed. First, the primary transfer was evaluated. We asked 10 people, 5 men and 5 women, to wash their hands with neutral pH soap, and then dry their hands with a specific towel. In the second experiment, the secondary transfer was evaluated. Five couples were asked to hold hands for 5 minutes. Then, they were asked to wash their hands with neutral pH soap, and dry them on a different towel each. Each experiment was performed 3 times each pair. As a control, an un- used piece of each towel was analysed, and saliva samples were taken from all participants, to perform genetic comparisons with the possible profiles found on the towels. DNA extracts were eluted out of the towels and profiles were evaluated through the amplification of MiniSTRs.

138 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P058 - ENHANCING THE SEXUAL ASSAULT WORKFLOW: DEVELOPMENT OF A RAPID MALE SCREENING ASSAY INCORPORATING MOLECULAR NON-MICROSCOPIC SPERM IDENTIFICATION Paris Volk1, Allison Holt2, Angela Chen2, Erin Hanson3, John Ballantyne1,3

1 University of Central Florida, Department of Chemistry, Orlando, USA 2 Thermo Fisher Scientific, Human Identification Research and Development, San Francisco, USA 3 University of Central Florida, National Center for Forensic Science, Orlando, USA

Sexual assault samples are among the most difficult sample types encountered by forensic laboratories. Typically, a sexual assault sample poses multiple challenges including small quantity of male DNA and a relatively high quantity of female DNA. Differential extraction procedures are time-consuming and labor-intensive, particularly with microscopic sperm identification. A rapid upfront screening or triage assay prior to use of differential extraction procedures could ensure that male DNA is present prior to the use of full DNA workflows. Previously reported rapid male screening assays do not provide a confirmation of whether sperm are present and therefore still require the use of microscopic sperm identification. This work was focused on an improved rapid male DNA screening assay with an upfront sperm identification using mRNA profiling. The rapid male screening assay consists of a brief lysis using only a small tip portion of a swab, to obtain both RNA (eluted) and DNA (extraction ‘waste’) followed by a one-step reverse transcription-high resolution melt assay for PRM2, a sperm-specific mRNA. Without additional purification, the DNA ‘waste’ fraction can be quantitated and used to obtain an upfront Y-STR profile of the sperm donor. The assay takes 30 min for lysis followed by ~2 hours for quantitation and sperm identification. Here, we demonstrate the specificity of the assay to detect male DNA in semen samples with no cross reactivity with other body fluids and no PRM2 detection in vasectomized males. RNA and DNA profiling results are obtained with as little as ~0.15 μl of semen in vaginal-semen admixtures. In all samples tested, Y-STR profiling results were obtained when male DNA was indicated based on quantitation results.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 139 P059 - ENVIRONMENTAL INFLUENCES ON POST MORTEM BRAIN MRNA TRANSCRIPTION Silviene Oliveira1, Raphael Bonadio1, Larissa Nunes1, Patrícia Moretti2, Aline Pic-Taylor1

1 University of Brasilia, Genetics and Morphology, Brasília, Brazil 2 University of Brasilia, Faculty of Healh, Brasília, Brazil

Studies have been shown that intracellular biomolecules continuously use energetic resources to maintain their function, even after the organismal death. Although most of the transcriptome is degrading after death, approximately 1 % is turned on. In this study we aimed to: (i) better understand the environmental influences in degradation and transcription after death; (ii) to identify brain transcripts with low environmental influences for use as estimators of post-mortem interval (PMI); and (iii) to identify the major pathways that turn on after death. We performed three corpse concealment simulations using C57BL/6jn mice: Exposed to air; Buried and Sub- merged in water. Brains were sampled in 24, 48, 72, 96 and 192 hours after death. The control group consisted of three newly deceased animals, which brains were extracted in the moment of death. The mouse brain transcriptome was evaluated using microarray, and the validations were done by qPCR. The enrichment analysis was performed to identify biological processes that could be possibly activated after death or associated with concealment types. Total RNA concentration decayed along the days of concealment and we suggest two molecular markers - F3 and Fabp7 - as estimators for postmortem interval. We observed 1279 down and upregulated transcription comparing the concealment groups and the control. From these, 63 were upregulated in all groups and 90 were exclusive for each environmental situation: 22 for buried, 30 for exposed and 38 for submerged groups. The majority of these upregulated transcripts are involved in ribosome biogenesis and peptide metabolic process. Our results show that each environment altered the transcriptome in its particular way and the data strongly suggest there is probably postmortem translation.

140 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P060 - EPIGENOME-WIDE ASSOCIATION STUDY FOR SUDDEN UNEXPECTED INFANT DEATH Masaki Hashiyada1, Masahiro Obayashi1, Tomohiro Matsumoto1, Sumitaka Yoshimura1, Atsushi Akane1

1 Kansai Medical University, Legal Medicine, Hirakata, Japan

Background and aims: The huge numbers of genome variants associated with sudden unexpected death have been found by the next generation sequencing (NGS) or massively parallel sequencing (MPS). However, these variants are just the “risk factors” based on the clinical patho- genetic variant database or the predictive algorithm, such as SIFT, Poly-Phen2 and Grantham. In this study, we applied the epigenome-wide association study for sudden unexpected infant death (SID) cases to get additional information to determine the cause of death. Material and methods: Genomic DNA was extracted from the septal cardiac muscle in autopsy cases of six infants (three SID cases, and each one case of myocarditis, asphyxia and Down syndrome) and an adult (intracerebral bleeding case). After bisulfite treatment, methylation profiles were measured using the Infinium MethylationEPIC BeadChip Kit and iSCAN system (Illumina). This can check over 860,000 methylation sites per sample at single-nucleotide resolution. Meth- ylation levels were compared among SID and other cases by GenomeStudio and Illumina Diff- Score software (Illumina) in four patterns. Top 50 increasing and decreasing methylation level sites were obtained. Results and discussion: In all four comparison patterns, SID cases showed a severe decrease in methylation level at cg05176970 and cg16032134 sites. The former site exists in NXN gene which is a novel thioredoxin family member involved in cell growth and differentiation. The latter site exists in ATP10A gene coding ATPase class V type 10A enzyme. In SID cases, these two genes expressed actively. Each of the other cases showed 20~30 sites with highly-changed methylation levels. Further analysis of more SID samples is required to determine the cause of death.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 141 P061 - ESTIMATION OF GENOTYPING ERRORS OF SSR MARKERS IN DOGS AND WOLVES Radka Štikarová1, Jakub Vašek1, Pavel Vejl1, Daniela Čílová1

1 Czech University of Life Sciences Prague, Department of Genetics and Breeding, Prague, Czech Republic

In the last years, many articles about dog and wolf hybridization in the wild were published. Lots of them differ in the type of used material, number, and type of samples, type of markers that were selected for identification purposes, etc. This article will discuss the estimation of genotyping errors in SSR markers on two types of biological material (buccal/excrement) in two populations (wolf/dog). Czechoslovakian wolfdog breed was used for the first group because it is FCI (Fédération Cynologique Internationale) recognized dog breed which is most phenotype and genotype relation to the wolf. This first group uses a total of 170 genotypes of this dog breed. The second group was formed from the Eurasian wolf breed, which consisted of 39 DNA samples. Mini-DogFiler was selected as a forensic identification microsatellite panel for this study. This panel was developed as a tool for animal identification from degraded DNA samples. The analysis showed very different results in genotyping error estimation. Buccal samples had a very low number of error occurrences. 0,09 error for one allele, 0,18 error on genotype and 2,17 error for genetic profile. These values were lower than the results that were published before. Presence of technical null allele for locus VGL2136 was recognised, but the cause wasn’t determined. Analysis of excrement samples affirms how is complicated find reliable results. For this type of samples is typical very fragmented DNA with low concentration. This fact leads to the occurrence of many of negative effects like an allelic dropout, zero allele or presence of PCR artefacts. Results confirm how are necessity of multiple isolate DNA from one sample and a multiple amplification of these samples for creating a consensus for each genotype.

142 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P062 - ESTIMATION OF THE NUMBER OF CONTRIBUTORS IN MIXTURE SAMPLES BY MITOCHONDRIAL DNA ANALYSES USING MASSIVELY PARALLEL SEQUENCING Hiroaki Nakanishi1,2, Koji Fujii3, Hiroaki Nakahara3, Natsuko Mizuno3, Kazumasa Sekiguchi3, Masanori Otani1, Katsumi Yoneyama2, Masaaki Hara2, Aya Takada2, Kazuyuki Saito1

1 Juntendo University School of Medicine, Forensic Medicine, Tokyo, Japan 2 Saitama Medical University, Forensic Medicine, Moroyama, Japan 3 National Research Institute of Police Science, Forensic Biology, Kashiwa, Japan

We evaluated whether the number of contributors in sample mixtures can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A-amplicon (positions 16,209–16,400) and B-amplicon (positions 30–284) in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trim- ming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for A- and B- amplicons using Microsoft Excel. The haplotype ratios were calculated by dividing the counted read number of the corresponding haplotype by the total sequence reads. To discriminate noise haplotypes, the threshold was set based on the results of single source samples. The haplotypes that were over the threshold (8.1 %) were defined as positive haplotype. The number of larger positive haplotypes in either of two amplicons was defined as the number of contributors. The number of contributors was correctly estimated from almost all mixed samples containing equal amounts DNA from 2–5 persons. In the mixed DNA samples of two or three persons, the minor components were detected down to the ratio of 10:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused an incorrect estimation of the number of contributors. Although this method still has room for improvement but is useful as it does not use complicated forensic mathematics.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 143 P063 - EUROPEAN VALIDATION OF A CANNABIS SATIVA 13-LOCUS STR MULTIPLEX KIT FOR GENETIC IDENTIFICATION: A PRELIMINARY STUDY Michele Di Nunzio1,2, Madeline G. Roman3, Rachel Houston3, Ciro Di Nunzio4, David Gangitano3, Carme Barrot Feixat1

1 University of Barcelona, Public Health, Barcelona, Spain 2 Biogem scarl, Forensic Genetics, Ariano Irpino, Italy 3 Sam Houston State University, Department of Forensic Science, Huntsville, USA 4 Magna Graciae University, Legal Medicine, Catanzaro, Italy

Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine and intoxicant. Traditionally, is divided into two main types: fiber type (hemp) and drug type (marijuana). Mari- juana differs from hemp by the presence of a high quantity of the psychoactive drug, Δ9-tetrahydrocannabinol. The development of a validated method using short tandem repeats (STRs) could serve as an intelligence tool to link cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13-loci STR multiplex method was developed, optimized, and validated by the Department of Forensic Science at Sam Houston State Univer- sity according to relevant ISFG and SWGDAM guidelines. The European community considers C.sativa plants illegals, even though its consumption is accepted in precise and limited places (Coffee Shops or Cannabis Clubs in Netherlands and Spain). However, there are different gaps in the legislation of some European countries. For instance, in Italy “weed” possession is decriminal- ized. Although, trafficking and sale are prohibited; possession of small quantities of “marijuana” is considered only a civil offense. In order to proceed with the kit evaluation and inter-laboratory validation, their DNA lab. sent us blind cannabis DNA samples of known genotypes. Blind DNA samples were analyzed in some countries’ laboratories with different sequencers and analysis conditions. In this article, the goals are: a) to demonstrate that 13-loci STR kit for C. sativa is robust enough and reproducible in all forensic laboratories and, b) to show the applicability of the STR system in association with Cannabis sativa cases for intelligence purposes to link multiple cases by means of genetic individualization or association of cannabis samples.

144 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P064 - EVALUATING THE POTENTIAL OF miRNA PROFILING IN VITREOUS HUMOR TO DETERMINE THE TIME OF DEATH Beatrice Corradini1, Milena Alù1, Simona Vecchio1, Enrico Silingardi1

1 Institute of Legal Medicine, Department of Biomedical- Metabolic and Neural Science, University of Modena and Reggio Emilia, Modena, Italy

The estimation of the moment of the day (day/night) when a death occured is of great importance in the field of forensic science and criminal investigation, leading to possible legal reper- cussions. Several methods are currently available, however they are affected by the lack of precision and reproducibility especially after long post-mortem periods. Vitreous humor is a highly stable matrix widely used in forensics but its molecular background has been only recently highlighted. MicroRNAs demonstrated to have a 24-hour light and dark cycle-dependent expression (circadian) in some specific human tissues. Here we tested the potential of microRNAs as “chronobiomarkers” to improve the estimation of the time of death. An initial screening was performed to deepen the microRNA signature of vitreous humor from 77 individuals who died at daytime and at nighttime with TaqMan Advanced miRNA cards assay. Among the microR- NAs tested, those with a differential expression between the two groups were further analysed with single Taqman Advanced assays to confirm the time-depent oscillating expression. Several challenges should be resolved to make microRNAs reliable biomarkers in post-mortem inventi- gations, such as the knowledge on the best normalization strategy in the qPCR assay, the body fluids specificity and their degradation kinetics under different environmental conditions. How- ever microRNAs display attractive characteristics to be used in the future as ancillary tool for the forensic pathologist in postmortem evaluation of the time and circumstances of death.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 145 P065 - EVALUATING THE VIABILITY OF OBTAINING DNA PROFILES FROM DNA ENCAPSULATED BETWEEN THE LAYERS OF COMPOSITE COUNTERFEIT BANKNOTES Ross Kwok1, Graham Williams1, David Kenny1

1 Staffordshire University, School of Law, Policing and Forensics, Stoke-on-Trent, United Kingdom

Banknote counterfeiting can potentially undermine the integrity of a currency, by eroding both public and retailer confidence in cash as a method of payment. In order to prevent this, banknote issuing authorities employ a range of overt and covert technologies, most in the form of banknote security features. The development, selection and deployment of such features, is an ongoing process undertaken jointly between the manufacturers of security features, banknote printers and banknote issuing authorities, i.e. Central Banks. This ongoing process ensures that the integrity of banknotes as a safe and secure means of payment is retained. While some counterfeit banknotes are seized by police at the production site or in storage, others are removed from circulation during banknote sorting operations, as part of the ‘cash cycle’. Counterfeit banknotes which are removed from circulation are inevitably contaminated, in terms of fingermarks and DNA acquired during handling by both criminals and non-criminals alike. However, encapsulated DNA recovered from between the layers of a composite banknote, is highly likely to belong to a person involved in the manufacturing process and is therefore of far greater evidential value. Such evidence has the potential to identify the perpetrator(s). This research evaluates the investigative potential of recovery and profiling of such encapsulated DNA, primarily with regard to specific counterfeit types. Accordingly, the goal of the research will be to establish an innovative and reliable method of extracting and profiling encapsulated DNA from counterfeit banknotes.

146 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P066 - EVALUATION OF DNA LEVELS RECOVERED FROM FORENSIC BONE SAMPLES THROUGH THE OPTIMIZATION OF A SEMI-AUTOMATED EXTRACTION METHOD Maria João Caldeira1,2, Ana Margarida Bento2, Nair Gouveia2, Pedro Brito2, Maria João Porto2

1 Aveiro University, Biology Department, Aveiro, Portugal 2 National Institute of Legal Medicine and Forensic Sciences, Forensics Genetic Service- Centre Branch, Coimbra, Portugal

A mass disaster presents adverse conditions that can lead to extensive carbonization, fragmentation and putrefaction making it difficult to obtain a good DNA profile for body identification. Often, hard tissues are the only feasible samples for extraction. Bones and teeth are sometimes also degraded, so extraction methods have to be chosen in order to recover as much genetic material as possible. DNA was extracted from eleven bone samples using PrepFiler BTA Extraction kit with the Auto- Mate Express with 20 and 50 μL of elution volume. Extracted DNA was quantified in a 7500 Re- al-Time PCR System with Quantifiler Trio. Amplifications were performed on the Veriti Thermal Cycler using PowerPlex Fusion 6C kit and GlobalFiler. The electrophoresis was carried out on a ABI 3500 and data analysis was done using GeneMapper ID-X. The objective was to compare extraction efficiency with different elution volumes, evaluating DNA recovery and quality of the obtained profiles, observing if the samples concentration alters the degradation index and/or the presence of inhibitors. The analyzed samples obtained an increase of the DNA levels with the 20 μL elution volume and the inhibition values did not change among the protocols, however some changes in the DI were observed. Undesirably, when the available DNA concentrations are low, the 20 μL protocol demands an increased number of extractions per sample as normally PCR reactions require as much as 15 μL input volume. This 20 μL method proved to be useful for difficult samples with low levels of DNA and high degradation, being able to increase recovered DNA concentration and obtain higher quality genetic profiles compared to the 50 μL protocol, increasing the efficiency in disaster victim identification cases.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 147 P067 - EVALUATION OF NEXT GENERATION mtGENOME SEQUENCING BASED ON DIFFERENT LIBRARY CONSTRUCTION PRINCIPLES Chi Zhang1

1 Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, Ministry of Public Security, Beijing, China

One of Next Generation Sequencing (NGS) strengths is to detect DNA Sequence variations, and now NGS has been widely interested by researchers in the field of forensic genetics. As one of the important genetic markers in forensic genetics, mitochondrial DNA (mtDNA) has the advantages of maternal inheritance, low recombination rate, high copy number and highly conservative coding region. MtDNA can be used for forensic human and species identification testing and ancient DNA analysis. There are two different methods to enrich the mtDNA library, one is PCR enrichment, the other is DNA probe enrichment. As to evaluate the applicability of the two different methods, we extracted DNA from hair, blood, buccal swabs and bones, divided into high DNA concentration group and low DNA concentration group, library constructed by two different methods above-mentioned. The mtDNA library which enriched by DNA probe can not be sequenced because of the low library concentration, even the high DNA templet concentration group. But the mtDNA library which enriched by PCR (based on The Precision ID mtDNA Whole Genome Panel, Thermo Fisher) can constructed the high concentration mtDNA library, which can be sequenced by Ion S5 XL, with average sequencing depth more than 3452.08X.

148 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P068 - EVALUATION OF STR PROFILES OF SINGLE TELOGEN HAIR USING PROBABILISTIC METHODS Sarah Aurora Heß1, Richard Jäger1

1 Hochschule Bonn-Rhein-Sieg University of Applied Sciences, Natural Sciences, Rheinbach, Germany

Single telogen hairs are rarely included in forensic STR analysis due to the minute amount and compromised integrity of their DNA which often result in incomplete profiles. Amount and integrity of DNA of single telogen hairs can be determined using qPCR assays which help choosing the optimal analytical strategy: Capillary electrophoresis (CE)-based standard protocols or an in- house developed next generation sequencing (NGS)-based multiplexed method called maSTR (mini-amplicon STR) assay. The latter uses very short amplicons and is particularly suited for degraded DNA. In such samples, CE-based analysis showed more allele drop-outs than the maSTR assay. However, the latter displayed a higher number of allele drop-ins, feigning mixed profiles. Those might be due to analytical errors of the NGS method or represent contaminations. Ac- cording to ISFG recommendations, probabilistic approaches should be applied in cases in which more than two drop-ins occur at more than two loci in one low-template DNA profile. To test in how far such approaches can improve the identification of the true genotype in cases of strong DNA degradation and analytical artefacts, NGS-derived STR profiles of single telogen hairs were evaluated on the basis of a fully continuous and a semicontinuous probabilistic model. In fully continuous models, a series of simulations provides for statistical weightings of all possible genotype constellations and the resolution of major and minor components. This increases the significance of the likelihood ratio compared to binary or semicontinuous models. On the basis of our investigations, we provide recommendations for DNA analysis of single telogen hairs.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 149 P069 - EVALUATION OF THE FORENSEQ® DNA SIGNATURE PREP KIT WITH SKELETAL REMAINS FROM MISSING PERSONS CASES Michelle Peck1, Šejla Idrizbegović1, Dyon Doensen1,2, René Huel1, Thomas Parsons1

1 International Commission on Missing Persons, Science and Technology, The Hague, Netherlands 2 University of Amsterdam, Institute for Interdisciplinary Studies, Amsterdam, Netherlands

The ForenSeq® DNA Signature Prep Kit holds promise for missing persons cases due to the number and variety of included markers. The STR markers maintain backwards compatibility with existing databases, while also providing sequence-based information. The SNP markers provide forward compatibility as the field moves toward SNPs, which are particularly suited for degraded DNA and more easily typed with massively parallel sequencing. Initial studies with bone extracts previously successful with CE-STR testing, revealed challenges inherent to forensic bone samples most likely due to the presence of inhibitors. The following modifications were explored to improve results: 1) purification of extracts with a 2X AMPpure XP bead ratio, 2) a modified PCR1 buffer (provided by Verogen), 3) library purification with 70 % EtOH:Tris-EDTA. The combined effect of these modifications resulted in a substantial increase in success rate, with the extract purification having the biggest impact on data recovery. Combined with the buffer and library purification modifications, full to partial results were obtained with a majority of samples that completely failed initial testing. Yet, due to some samples still showing limited success, further studies are needed on a wider range of bone sample types. It is critical to establish appropriate thresholds for low input samples to account for stochastic effects. Thresholds specific to the overall performance of the locus were evaluated resulting in the retention of valuable data without increasing risk of false homozygote calls. The increased data content from the DNA Signature Prep kit profiles has proven beneficial in several missing persons cases, and strategies for implementation of this method into routine missing persons cases will be explored.

150 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P070 - EVALUATION OF VAGINAL mRNA MARKERS IN FERTILE AND POSTMENOPAUSAL WOMEN: A GEFI COLLABORATIVE STUDY Carlo Robino1, Elena Chierto1, Federica Alessandrini2, Carla Bini3, Eugenia Carnevali4, Matteo Fabbri5, Paolo Fattorini6, Pierangela Grignani7, Francesca Scarnicci8, Pamela Tozzo9, Andrea Verzeletti10, Susi Pelotti3, Loredana Buscemi2

1 Università di Torino, Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Torino, Italy 2 Università Politecnica delle Marche, Dipartimento di Scienze Biomediche e Sanità Pubblica, Ancona, Italy 3 Università di Bologna, Dipartimento di Scienze mediche e chirurgiche, Bologna, Italy 4 Università di Perugia sede di Terni, Dipartimento di Medicina Clinica e Sperimentale, Terni, Italy 5 Università di Ferrara, Dipartimento di Scienza biomediche e chirurgico specialistiche, Ferrara, Italy 6 Università di Trieste, Dipartimento di Scienze mediche- chirurgiche e della salute, Trieste, Italy 7 Università di Pavia, Dipartimento di Sanità Pubblica- Medicina Sperimentale e Forense, Pavia, Italy 8 Università Cattolica del Sacro Cuore di Roma, Istituto di Sanità Pubblica, Roma, Italy 9 Dipartimento di Università di Padova, Dipartimento di Medicina Molecolare, Padova, Italy 10 Università di Brescia, Dipartimento di Specialità Medico-Chirurgiche- Scienze Radiologiche e Sanità Pubblica, Brescia, Italy

In the past few years mRNA profiling has emerged as a powerful tool for body fluid identification in criminal investigations. Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Vaginal mucosa is a highly dynamic tissue undergoing several morphological and physiological modifications over a lifetime in connection with hormonal modulation and ageing. However, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups. In this study, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1), which was previously implemented in GeFI (Italian working group of ISFG) laboratories [1], was tested in a collection of vaginal swabs obtained from fertile (n=84) and postmenopausal (n=55) female volunteer donors. Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa “observed” in the tested sample according to a previously defined scoring protocol [1]) b) peak heights ratio between vaginal-specific markers and housekeeping genes observed in capillary electrophoresis experiments. Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated. [1] Carnevali E et al. A GEFI collaborative exercise on DNA/RNA co-analysis and mRNA profiling Interpretation. Forensic Sci Int Genet Suppl Ser. 2017;6:e18-e20.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 151 P071 - EVERYDAY CHALLENGES OF DATA INTERPRETATION IN CAPILLARY ELECTROPHORESIS RESOLVED USING APPLIED BIOSYSTEM DATA COLLECTION & GENEMAPPER ID-X ANALYSIS SW Priti Sabadra1, Jaime Brachold1, Ariana Wheaton1, Angela Lackey1

1 Thermo Fisher Scientific, Human Identification, S. San Francisco, USA

For over 25 years, Applied Biosystems has used customer feedback to help shape technology to meet the ever-evolving needs of the forensic community. In the last few years, as labs worked to implement expanded multiplex kits, a new set of data interpretation challenges arose. Labs, more than ever before, need ways to decrease analysis time, increase confidence in resultant data, and more efficiently process samples and data. In response to this need, the newest versions of Applied Biosystems 3500 Data Collection and GeneMapper ID-X were developed. Up to this point, only a high level introduction to the 3500 DC v4.0 and GeneMapper ID-X v1.6 improvements has been discussed. Now let’s take a look under the hood at the technical processes and functions behind pull-up reduction, off- scale data recovery, and signal optimization that will improve data interpretation by decreasing analysis time and enhancing efficiency.

152 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P072 - EXPANDING THE KNOWLEDGE OF TRI-ALLELIC PATTERN AT AUTOSOMAL STR LOCI IN CHINESE POPULATION Qinrui Yang1, Chengchen Shao1, Qiqun Tang2, Jianhui Xie1

1 Fudan University, Department of Forensic Medicine- School of Basic Medical Sciences, Shanghai, China 2 Fudan University, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai, China

The identification of tri-allelic pattern could elevate the discrimination power and lower the risk of false exclusion during forensic practice. Concerns have been raised that the omission, mis- judgement, and inappropriate interpretation of tri-allelic locus could impair the weight of evidence in specific cases. This research aims at comprehensively analyzing tri-allelic pattern at autosomal STR loci. As the generation of Type 1 and Type 2 attributes to distinct mechanisms, the study on two types of pattern was carried out respectively based on a pool of Chinese population. The occurrence of tri-allelic genotypes at CODIS STR loci (including CSF1PO, FGA, TH01, Vwa, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11) was investigated. New allelic combinations were reported. The incidence of Type 1 showed correlation with the property of STR loci. On the other hand, different traits were observed on the incidence of Type 2. An abnormally higher frequency was obtained at TPOX in Chinese population. The identical form of configuration into Chr2 of the extra copy of Type 2 at TPOX in Chinese population might explain their shared origin and expound its higher frequency among populations. Type 2 at other STR loci is more likely to be produced spontaneously in populations, while stable inheritance could hardly be expected. Further, family cases concerning tri-allelic genotypes were reported. Allele transmission in Type 1 and Type 2 was generalized, and the inheritance pattern was further indicated. Comparison was presented with other populations. Altogether, we would like to give a better understanding of tri-allelic patterns at autosomal STR loci in Chinese population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 153 P073 - EXPLORATION OF FTIR-ATR SPECTROSCOPY COMBINED WITH DATA MANIPULATION TO PREDICT DNA PRESERVATION IN SKELETAL REMAINS Tamara Leskovar1, Irena Zupanič Pajnič2, Ivan Jerman3, Matija Črešnar1

1 University of Ljubljana, Faculty of Arts- Department of Archaeology, Ljubljana, Slovenia 2 University of Ljubljana, Medical Faculty, Institute of Forensic Medicine, Ljubljana, Slovenia 3 National Institute of Chemisty, National Institute of Chemisty, Ljubljana, Slovenia

Skeletal remains are commonly subjected to various analyses, including DNA. As they are exposed to taphonomical processes, their chemo-physical structure is altered. The success and integrity of the DNA analyses thus depend on the preservation state of the sample. Several techniques were tested as pre-screening methods for the evaluation of DNA preservation in skeletal remains. Although some show potential, they are financially demanding and time-consuming, with limited predictive power. In this study, ATR-FTIR spectroscopy with further data manipulation was explored for the purpose, hypothesising that spectra of samples of different origin and preservation differ enough to be classified according to their potential for successful DNA extraction. 138 human bones and teeth originating from archaeological, WWII or forensic context were used. Samples were cleaned and powdered following established methodological procedures for DNA extraction. DNA was extracted and quantified, separating samples into 4 arbitrary classes (1=worst – 4=best preservation). The remaining powder was analysed with ATR-FTIR and spectra manipulated to extract chemo-physical information. Combining acquired data, statistical analyses, machine learning, and predictions were performed. 110 samples were used for the analyses and 28 for testing the procedures. Best results were achieved using the random forest learning algorithm on normalised and 2nd derivative spectra, and 8 highest ranking peak ratios. Even though overlapping remains, obtained results indicate that combined FTIR-ATR and statistical analyses have high potential as a pre-screening method for the evaluation of DNA preservation in the skeletal remains, especially when skeletal element and chronological age of the sample are acknowledged.

154 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P074 - EXPLORING OF RARE DIFFERENCES IN mtGENOMES BETWEEN MZ TWINS USING MASSIVELY PARALLEL SEQUENCING Tianyue Ming1, Mingrui Zheng1, Yanping Zhou1, Yiping Hou1, Zheng Wang1

1 Sichuan University, Institute of Forensic Medicine, Chengdu, China

Compared with nuclear DNA, fewer DNA repair mechanisms in mitochondria and lack of proof- reading capabilities in the mtDNA polymerase help introduce more variability between MZ twins. In our previous study, we used ultra-deep mtGenome sequencing to characterize point heteroplasmy and nucleotide variant in blood samples of MZ twins. In the present study, we characterize minor differences of mtGenomes in saliva and hairshaft samples from six sets of MZ twins using the Precision ID mtDNA Whole Genome Panel, Ion S5 XL system, and Converge Software. Additionally, the effectiveness of different tissue samples for differentiating between MZ twins was evaluated. Point heteroplasmies were observed in all sets of MZ twins regardless of sample type. Overall, more variants were observed in the mtGenome from hair shaft samples than that from blood and saliva samples. The results of this study further support that the mtGe- nome analysis could be used to distinguish MZ twins from each other.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 155 P075 - FLANKLY, IS IT WORTH IT? Laurence Devesse1, David Ballard1, Lucinda Davenport1, Denise Syndercombe Court1

1 King’s College London, King’s Forensics, London, United Kingdom

The application of massively parallel sequencing (MPS) to forensic genetics has led to improvements in multiple aspects of DNA analysis, however additional complexities are concurrently associated with these advances. In relation to STR analysis, the move to assign alleles using sequence rather than length-based methodologies has highlighted the extent to which previous allelic variation was masked. In this work, a series of samples (n=1000) from five different population groups were genotyped using the MiSeq FGx™ Forensic Genomics System. Sequence variation has been characterised both within and outside STR repeat regions. At certain loci, the increase in allelic diversity when considering flanking region variation is significant – notably, D16S539 which shows very little repeat region variation but has a 100 % increase in the number of distinct alleles observed when accounting for flanking region variants. However, 16 out the 27 loci studied show little to no variation outside of the repeat regions. Of the remaining STRs, D12S391 is the most polymorphic, where the number of alleles increases from 25 length-based to 98 sequence-based alleles, yet only 12 % of this increase is provided by flanking region variants. The challenges associated with sequence-based allele nomenclature expand markedly when considering flanking regions, bringing into question the value of these regions for forensic casework – is the effort worth it? This presentation will illustrate how flanking region variation differs across markers and populations, as well as provide examples of complex relationship cases and how useful (or not) both repeat region and flanking region sequence variants can be.

156 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P076 - FORENSIC IDENTIFICATION OF MATERIAL FROM HUMAN ANUS BASED ON METAPOPULATION STUDIES OF THE BACTERIAL 16S RNA GENE Jolanta Powierska-Czarny1, Agnieszka Piotrowska-Cyplik2, Łukasz Kaczorowski1, Monika Antczak1, Magdalena Chrostowska1, Aleksandra Górka1, Joanna Idkowiak1, Bartosz Hornik1, Paweł Cyplik3, Jakub Czarny1

1 Institut of Forensic Genetics, Department od Forensic Genetics, Bydgoszcz, Poland 2 Poznan University of Life Sciences, Institute of Food Technology of Plant Origin, Poznań, Poland 3 Poznan University of Life Sciences, Department of Biotechnology and Food Microbiology, Poznań, Poland

In some types of sexual crimes, it is necessary to extend the range of standard tests for the identification of biological material with the identification of vaginal, preputial or anal secretions. In case of the identification of material from the anus and faeces, the identification of faecal bacteria characteristic for this environment is the preferred method. The study involved 21 swabs from victims of sexual offenses in which no semen and genetic material of other people (sexual partners) were found. The DNA was isolated using the QIAsympho- ny DNA Investigator Kit (Qiagen) and a QIASymphony workstation. Regions 2, 3, 4, 5, 6, 7, 8 and 9 of the 16S RNA gene were amplified using the Ion 16S™ Metagenomics Kit (Life Technologies). Libraries were prepared using the Ion Plus Fragment Library Kit (Life Technologies) and library amplification was carried out with the Ion PGM™ Hi-Q™ View OT2 Kit using the Ion OneTouch™ 2 System (Life Technologies). Sequencing was carried out based on an Ion Personal Genome Machine™ (PGM™) System (Life Technologies) using the Ion PGM™ Kit Hi-Q™ View Sequencing Kit (Life Technologies) and Ion 316™ Chip Kit v2 BC (Life Technologies). A group of species characteristic for faecal microflora was identified among the identified bacteria and comparison of results with the analysis of other human body environments indicates that metapopulation analysis can be successfully used as a tool to identify biological material from the anus in sexual crimes.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 157 P077 - FORENSIC SCIENCE & HUMAN MIGRATION: THE ROLE OF FORENSIC GENETICS Emma Johnston1

1 De Montfort University, The Leicester School of Pharmacy, Leicester, United Kingdom

Many migrants perish on dangerous journeys in search of a better life around the world. Howev- er, little is being done to identify these individuals. Forensic science has successfully been used to identify individuals following mass-casualty incidents such as 9/11, the Boxing Day tsunami and the Balkans war. In these cases and many others, forensic scientists have given evidence in international tribunals and helped to provide answers to families searching for their loved ones. The international forensic science community is responding to this humanitarian crisis and at- tempting to identify decedent migrants. However, due to the complex nature of the migrant situation there are practical limitations to some conventional identification methods such as dental records and DNA profiling. Antemortem data can be difficult to gather. DNA profiling is still the gold standard technology for human identification. However, the limitations of conventional forensic identification methods in the migrant context may mean that innovative, new scientific techniques are required. This poster will discuss current efforts to aid the forensic identification of migrants using forensic genetics and potential future uses. Key questions and issues such as the use of databases and ethics will be considered.

158 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P078 - GENDER TYPING OF HIGHLY FRAGMENTED HUMAN DNA SAMPLES Kijeong Kim1, Kyungyong Kim1

1 Ancient Human DNA Institute, College of Medicine, Chungang University, Seoul, Republic of Korea

The determination of human gender with fresh human DNA is very easy and rarely difficulty to do with it. But it has been well known to be very difficult to confirm the gender of severely degraded human DNA. The dropout of some alleles due to different amount in fragmented and severely degraded small quantity DNA seems to be very serious problem to type the gender. To rule out the genotyping errors, many studies require PCR amplification to solve this replication correctly. Usually the limiting factors are small amount and degraded level of human DNA samples. Here we want to report our study to verify the gender type with a real-time PCR-based amelogenin Y (amely). We introduced an allele dropout estimation model in an amel-based gender typing. The gender of all the degraded human DNA samples was confirmed by sex-determination region Y marker amplification. We also suggest this model as a securing amely allele dropout-safe method from degraded inhibitory DNA samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 159 P079 - GENETIC AND CHROMOSOMAL VARIATIONS CAUSED INCONSISTENCIES IN TWO PARENTAL TESTS Chen Li1, Yifan Li1, Li Jia2, Chong Chen2, Shi Yan2, Chuguang Chen1, Yacheng Liu2, He Ren3

1 Beijing Microread Genetics Co.- Ltd, Beijing Microread Genetics Co.- Ltd, Beijing, China 2 Beijing Tongda Shoucheng Insitute of Forensic Science, Beijing Tongda Shoucheng Insitute of Forensic Science, Beijing, China 3 Beijing Police College, Criminal Science and Technology, Beijing, China

Potential erroneous parental exclusions are possible to be found in trio parentage tests due to gene mutations or chromosomal variation. Here, we report two special cases where inconsistency between children and the fathers were observed at FGA locus. Regular additional STR tests were performed and no more inconsistency was observed in the first case. Through the following TA cloning and sequencing, the first case turned to be a two-step mutation between child and father. In the second case, however, one more inconsistency was found in the additional test at D4S2366. Both FGA and D4S2366 was on Chr.4 and homozygous, which led to a possibility of chromosomal variation. To prove this hypothesis, seven randomly selected STR-loci on Chromosome 4 were further analyzed and showed similar result, which indicated a possible maternal uniparental disomy (UPD) in the child with normal phenotype. This study emphasizes gene or chromosomal variations may mislead parentage test, especially variations like UPD that are relatively unfamiliar to investigators. If all the inconsistent loci are on the same chromosome, investigators should take UPD into consideration, and further tests, like chromosomal-specific STR-typing, should be applied to prevent pseudo-exclusions.

160 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P080 - GENETIC IDENTIFICATION OF A COMMUNISM REGIME VICTIM: THE STORY OF 70 YEARS OLD TENT TARPAULIN Simona Dalihodova1, Jitka Votrubova1, Daniel Vanek1,2,3

1 Forensic DNA Service, DNA laboratory, Prague, Czech Republic 2 Charles University in Prague, 2nd Faculty of Medicine, Prague, Czech Republic 3 Institute of Legal medicine, Bulovka Hospital, DNA laboratory, Prague, Czech Republic

In 1948 the government of the Czechoslovakia was violently taken over by the communists. The squad of Junak – Czechoslovak Scounting from Zelezný Brod (town in the north of the Czech Republic) did not want to submit to this regime and decided to leave Czechoslovakia to keep their traditions abroad. In the night of 24th July 1949, seven young people who decided to es- cape from their home land, were surrounded in a Jizera Mountains camp by the members of StB (State Security) and SNB (State National Security) and mercilessly gunned down. 70 years later we examined a blooded tent tarpaulin from the crime scene with the aim to perform DNA analysis of the blood stains and make a comparison with living relatives to identify the dead person that was wrapped in the tent tarpaulin.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 161 P081 - GENETIC STRUCTURE AND CONSERVATION STATUS OF THE BLUE SHARK BASED ON THE MITOCHONDRIAL D-LOOP REGION Amanda Bitencourt1, Dayse Silva2, Elizeu Fagundes Carvalho2, Silvia Martins1, Cesar Amaral2

1 Universidade do Estado do Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 2 Universidade do Estado do Rio de Janeiro, Departamento de Ecologia - Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil

The Elasmobranchii comprises the diverse and important group of sharks and rays. The group includes some of the ocean’s largest predatory fishes, being commercially overexploited due to unsustainable fishing activities for their meat and fins. Overfishing has resulted in significant population declines and several species are now considered under high threat with about 93 % of its nominal species included in the IUCN Red List. Among these threatened species, the blue shark (Prionace glauca) is one of the most abundant species of epipelagic zone (0–200 m) being widely distributed in temperate and tropical oceans. The specie is known by its great skill to perform large migrations influenced mainly due to the seasonal temperature variations of the oceans, better conditions for reproduction, and according to the availability of prey. Aiming to study aspects of the species conservation, the purpose of this study is analyze the population structure of the species based on newly determined and previously published D-Loop sequences. Molecular data has provided important information about threatened shark species, allowing the management of natural stocks. Population genetics and connectivity data are now available and play an important role on establishing conservation policies. However, despite the ecological, commercial and conservation importance of these species, the literature including samples from the South Atlantic waters is still scarce. The present study is based on several P. glauca individuals collected along the Brazilian coastal waters and commonly commercialized within Brazilian fish markets. Keywords: Prionace glauca; mitochondrial D-Loop; sharks.

162 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P082 - GENETIC TAPHONOMY: EVALUATION OF QUALITY AND QUANTITY OF DNA FROM SKELETAL REMAINS Joanna Arciszewska1, Sandra Cytacka1, Maria Szargut1, Grażyna Zielińska1, Katarzyna Jałowińska1, Andrzej Ossowski1

1 Pomeranian Medical University, Department of Forensic Genetics, Szczecin, Poland

The primary task for forensic scientists is to obtain good quality DNA which is essential in genetic profiling process. An important aspect of laboratory work with degraded material is the selection of a sample in which the highest concentration of DNA could be found. The study was based on the assessment of the mean concentration of DNA in selected parts of the skeleton. The research was carried out based on skeletal remains of people who died in years of 1939–1945. Fragments of the petrus bone, tooth, vertebra, rib, pelvis, humerus, radius, ulna, femur, tibia, fibula, phalanx were cleaned mechanically and chemically. Afterward, they were de- contaminated under a UV lamp and ground in liquid nitrogen. The prepared bone powder was subjected to isolation of the genetic material using the PrepFiler BTA Forensic DNA Extraction Kit, the process was repeated three times for each sample. The next steps were real-time PCR, DNA amplification and capillary electrophoresis. Evaluation of the purity of the isolate was assessed during the analysis of STR profiles. Statistical analysis was prepared from the obtained results. Preliminary studies, which were carried out on skeletal remains of five individuals, were preformed, the results are as follows: high DNA concentration in the sample occurs in the teeth (0,12958 ng/μl), successively in the petrus bone (0,09026 ng/μl) and first phalange (0,01818 ng/ μl). The average concentration of DNA in femur, commonly used for the purpose of personal identification in mass disasters, was (0,00208 ng/μl). The results of the study will impact the methodology of the human identification process in both mass disasters and ancient DNA studies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 163 P083 - GIVING THEM BACK THEIR NAMES AND FACES. DNA ANALYSES AND MATERIAL CULTURE STUDIES OF UNDOCUMENTED MIGRANTS FROM CENTRAL AMERICA AND MEXICO TO TEXAS Kate Spradley1, Marek Edward Jasinski2,3, Joanna Arciszewska4, Grażyna Zielińska4, Maria Szargut4, Andrzej Ossowski4

1 Texas State University, Department of Anthropology, San Marcos, USA 2 Norwegian University of Science and Technology, Department of Historical Studies, Trondheim, Norway 3 Falstad Centre, Falstad Centre, Ekne, Norway 4 Pomeranian Medical University in Szczecin, Department of Forensic Genetics, Szczecin, Poland

Since the mid-1990s, 7,000 migrants have died along the US/Mexico border and over 3,000 of these deaths occurred in Texas, the majority remain unidentified. There are no federal regulations regarding unidentified deaths in the United States, leaving responsibility of identification to each state. Texas law requires DNA sample submission to a CODIS laboratory. Unfortunately, in South Texas, many jurisdictions without medical examiner services buried these presumed migrant remains with little investigation and no DNA sampling, leaving an unknown number of migrants without the possibility of identification. Exhumation and identification efforts are now taking place by the Forensic Anthropology Center at Texas State (FACTS). A collaborative project was initiated through Falstad Center in Norway, Norwegian University of Science and Technology and Falstad Center, Pomeranian Medical University in Poland, and FACTS in United States. One objective of the collaboration is to provide positive identification to these migrants, explore cultural concepts of postmortem human dignity, as well as utilize genetic information to investigate/explore the genetic structure, geographic origin and phenotype information. Left, fifth metatarsal bone from 20 unidentified presumed migrants exhumed from the Sacred Heart Burial Park in Falfurrias, Texas were sent to the Department of Genetics PUM. The prepared bone was subjected to isolation of the genetic material using the PrepFiler BTA Forensic DNA Ex- traction Kit. The next steps were real-time PCR, DNA amplification and capillary electrophoresis. The results of these analyses will be discussed within the context of forensic identification and migrant’s identity.

164 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P084 - HIGH RESOLUTION MELTING ANALYSIS (HRM) BASED ON 16SrRNA AS A TOOL FOR PERSONAL IDENTIFICATION WITH THE HUMAN ORAL MICROBIOME Shuangshuang Wang1, Feng Song1, Yanyun Wang2, Bowen Xie1, Yun Huang1, Haibo Luo1

1 West China School of Basic Medical Sciences & Forensic Medicine- Sichuan University, Forensic Genetics, Chengdu, China 2 West China Second University Hospital- Sichuan University, Laboratory of Molecular Translational Medicine- Center for Translational Medicine- Key Laboratory of Birth Defects and Related Diseases of Women and Children Sichuan University- Ministry of Education, Chengdu, China

Personal identification plays an important role in the forensic practice. The conventional methods of personal identification including STR, SNP and InDel are focusing on the molecular characteristics of the human cell. Recently, researchers pay attention to human microbiome by the reason that the human microbiome is rich in amount and variable among people. The purpose of this study is to apply the human oral microbiome to forensic personal identification. We designed one general primer pair: 518F&806R, and five specific primer pairs: Streptococcus, Veillonella, Fusobacterium, Actinomycetes, Lactobacillus, which all targeted the 16SrRNA. We conducted the high resolution melting analysis (HRM) with the six primer pairs. The results indicated that oral microbiome from different people could be distinguished by using HRM based on 16SrRNA. This study showed that human oral microbiome could be a promising marker for the forensic personal identification application.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 165 P085 - HOW LONG CAN DNA BE SUCCESSFULLY INVESTIGATED WHEN SAMPLED POSTMORTEM FROM ANIMALS SLAUGHTERED BY PREDATORS? Jan-Hendrik Modrow1, Thorsten Schwark2, Niklas Nuss1, Nele Meyer1, Jamie Hempel1, Nicole von Wurmb-Schwark3

1 ForGen GmbH, Animal Forensics, Hamburg, Germany 2 Laboratoire national de santé, Département Médecine Légale, Dudelange, Luxembourg 3 ForGen GmbH, Forensic Genetics, Hamburg, Germany

The wolf is back in Europe. Many people welcome this and see its return as a sign of an intact and original nature. However, instead of primarily feeding on wild animals, and thus regulating the game population, the wolf also preys on farm animals and snatches sheep, horses, llamas, etc. DNA swabs are routinely taken from killed animals to clarify whether the culprit was a wolf, and, if so, to further individualize the animal. In many places the opinion prevails that securing evidence is no longer promising after 24 or 48 hours at the latest; this is based on the publication of a German working group. Consequently, in many cases, after this time has elapsed, no more samples are secured, sometimes causing a considerable financial loss for the owners of the killed animals. From a forensic point of view, however, this view must be strongly doubted.To refute the results of the aforementioned study, we conducted an experiment in which dogs chewed on animal extremities (with fur; samples of animals were provided by local hunters). After a defined period, the animal parts were taken from the dogs, and were laid out in the woods. Again, after defined intervals, swabs were taken, and genetic analyses to detect Canidae specific DNA were performed. Our results show that successful DNA typing is possible even after much longer time periods than 24 hours. As expected, the results were influenced by several other factors besides the postmortem interval, such as the temperature. The experiments and the results will be presented in detail. This study shows that DNA sampling in cases in which animals are presumably killed by a wolf should not be based on the PMI alone. These cases should be treated individually regarding all circumstances.

166 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P086 - HOW MANY DNA ANALYSES ARE PERFORMED ON ADULT SEXUAL ASSAULT VICTIMS IN MILAN (ITALY)? A TEN-YEAR REVIEW Andrea Piccinini1,2, Paolo Bailo1, Giulia Vignali3, Giussy Barbara2, Giuseppe Gennari4, Domenico Di Candia3, Valentina Albertini2, Alessandra Kustermann2

1 Forensic Genetics Laboratory, Department of Biomedical Sciences for Health - Università degli Studi di Milano, Milano, Italy 2 Department of Women’s and Children’s Health and Service for Sexual and Domestic Violence SVSeD, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy 3 Institute of Legal Medicine, Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milano, Italy 4 The Court of Milan, Tribunale di Milano - Via San Barnaba 50, Milano, Italy

The aim of this study is to evaluate the actual use of serological/DNA analyses in the investigations carried out on adult victims of sexual violence in Italy during the years 2006–2015. The victims were assisted in the largest Italian rape center, in Milan (Soccorso Violenza Sessuale e Domestica – SVSeD - Service for Sexual and Domestic Violence). The total number of sexual violence victims examined during the years 2006–2015 (adults and minors) was 3521, in 1697 of which biological evidence had been collected, while the number of adult victims (>18 y.o.) examined was 2300, 1224 of which with biological evidence collected. Biological evidence was collected from the victims’ bodies using two swabs in five anogenital areas (labia maiora, labia minora, perineum, perianal and anal/rectum regions) and two swabs in all other skin areas suggested by the victims as areas of possible contact (double swab technique). Clothes were also collected on a case by case basis for the search of biological stains. Despite the proper collection, handling and chain of custody for all the swabs/items collected, serological/DNA analyses were requested in 86 cases out of 1211 only (7,10 %). This percentage dropped to 1,90 % when considering adolescent victims (13–19 y.o.). It remains unclear the reason why Italian Magistrates make little use of the powerful tool of DNA analyses in sexual assault cases. Legal and procedural aspects are therefore also discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 167 P087 - IDENTIFICATION OF CETACEAN SPECIES IN FORENSIC CASES Andrés Lopez-Oceja1, Xabier Lekube2, Ruiz Leire3, Jose Antonio Mujika-Alustiza4, Maite Alvarez- Alvarez5, Marian M. De Pancorbo1

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 University of the Basque Country, Biscay Bay Environmental Biospecimen Bank. Research Centre for Experimental Marine Biology and Biotechnology. PiE-UPV/EHU., Plentzia, Spain 3 AMBAR Elkartea, Ambar-Elkartea, Plentzia, Spain 4 University of the Basque Country, Geography Prehistory and Archaeology, Vitoria-Gasteiz, Spain 5 University of the Basque Country, SGiker Banco ADN, Vitoria-Gasteiz, Spain

Dolphins and other cetacean are protected species whose capture is illegal in many countries. Despite the high mercury content accumulated by these animals, occasional episodes of illegal fishing occur to supply certain black markets with dolphin meat. When fishing authority inspec- tions detect meat portions presumably of cetacean origin, it is particularly necessary to identify the species before taking the case to court. The cytochrome b (cyt b) gene of mitochondrial DNA has been used extensively to identify species. In forensic cases, due to the degradation of the samples, short amplicons are preferable. The universal primers cyt b L15601 and H15748, previously tested in 63 species (López-Oceja et al., FSI Genet, 2016 Jul; 23: 159–165), have been used in this work on individuals of seven species of cetaceans: Balaenoptera physalus, Delphi- nus delphis, Globicephala melas, Phocoena phocoena, Stenella coeruleoalba, Tursiops truncates and Ziphius cavirostris. These universal primers, with a short amplicon of 148 bp, have the advantage of identifying postmortem samples even when showing obvious signs of degradation. Our results showed that all tested species were correctly identified, except S. coeruleoalba and S. clymene. This is because S. clymene arose by hybrid speciation between S. coeruleoalba and S. longirostris. Therefore, their identification requires the analysis of both mitochondrial DNA and nuclear DNA (Amaral et al., Plos One 2014: 9 (1): e83645). In summary, the identification of illegal- ly obtained cetacean meat can be easily performed in forensic laboratories by analyzing a short fragment of the mitochondrial cyt b in all the studied species except in S. coeruleoalba and S. clymene and even when the remains were in an advanced stage of degradation.

168 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P088 - IDENTIFICATION OF VAGINAL SECRETIONS BASED ON METAPOPULATION STUDIES OF THE BACTERIAL 16S RNA GENE Jolanta Powierska-Czarny1, Agnieszka Piotrowska-Cyplik2, Bartosz Hornik1, Łukasz Kaczorowski1, Joanna Idkowiak1, Monika Antczak1, Aleksandra Górka1, Paweł Cyplik3, Jakub Czarny1, Magdalena Chrostowska1

In case of sexual crimes, it is often necessary to investigate whether the disclosed female genetic material on the body and clothing of a suspect particularly originates from a vagina. There are basically two approaches to identification of vaginal secretions: micro RNA analysis and micro- biological analyses. We present the results of a metapopulation analysis of 25 vaginal swabs sampled from victims of sexual offenses in which no semen and male genetic material were found. The DNA was isolated using the QIAsymphony DNA Investigator Kit (Qiagen) and a QIASymphony workstation. Regions 2, 3, 4, 5, 6, 7, 8 and 9 of the 16S RNA gene were amplified using the Ion 16S™ Metage- nomics Kit. The libraries were prepared using the Ion Plus Fragment Library Kit and the Ion PGM™ Hi-Q™ View OT2 Kit. The sequencing was carried out using the Ion Personal Genome Machine™ (PG™) System with the Ion PGM™ Hi-Q™ View Sequencing Kit and the Ion 316™ Chip Kit v2 BC (all from Life Technologies). The sequencing data was processed using CLC Genomic Workbench 8.5 and CLC Microbial Genomics Module 1.2. (Qiagen, USA). The study results indicate that standard DNA isolation, which is used for the analysis of forensic traces, allows to obtain the genetic material of Gram+ and Gram- bacteria. Although the use of enzymatic lysis of bacterial cell walls increases the efficiency of bacterial DNA isolation, this modification does not seem to be necessary for the identification of bacteria. Comparison of the obtained results with the analyses of other environments allows for the selection of species characteristic for the female vaginal secretions. The presented results indicate that metapopulation analysis of the bacterial 16S RNA gene may be a useful tool for identification of vaginal secretions.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 169 P089 - IMPLEMENTATION OF THE PRECISION ID mtDNA WHOLE GENOME PANEL FOR FORENSIC CASE WORK: HOW TO HANDLE INCONSISTENT RESULTS Anders Buchard1, Vania Pereira1, Claus Boersting1, Eszter Rockenbauer1

1 Section of Forensic Genetics, Department of Forensic Medicine, Copenhagen, Denmark

The Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific) is currently being validated for forensic genetic investigations in our ISO 17025 accredited laboratory. For the validation study, 107 samples from Danes with known whole genome mtDNA profiles were selected. All samples were previously sequenced using the MiSeq platform (Illumina). Here, the samples were sequenced in duplicates using the IonTorrent PGM™ platform and once using the Ion S5™ System (Thermo Fisher Scientific). The sequencing data was processed with the Torrent Vari- ant Caller and Converge HID GenoTyper v2.1 plugins using default settings. Additional criteria (minimum read depth: 100; maximum noise: 7 %; heteroplasmy call: >15 %) were imposed on the data prior to comparison of mtDNA profiles. Inconsistencies were observed between 1) the duplicate typings on the IonTorrent PGM, 2) the Ion PGM and the Ion S5, 3) the Ion Torrent and the MiSeq platforms, and 4) the haplotypes generated by the different analysis software on the same sequencing output. Most (~80 %) of the inconsistencies were found in the homopoly- meric regions 302–315 and 16180–16193. These regions will not be considered for case work. However, the remaining inconsistencies were identified as presence of a variant in one profile and absence of the variant in the second profile. Thus, the observations were most likely not due to heteroplasmy but uncertainty of the base calling. This raises concerns and calls for more stringent analysis criteria. It also underlines the need for duplicate or even triplicate typing of the samples. The inconsistencies caused by different analysis software or different instrumentation are a particular problem for generation of accurate databases and for the implementation process.

170 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P090 - IMPROVED IDENTIFICATION OF HUMAN BONE REMAINS BY THE INTRODUCTION OF A SNP ANALYSIS SYSTEM Sohee Cho1, Moon Young Kim1, Soong Deok Lee1,2

1 Seoul National University College of Medicine, Institute of Forensic and Anthropological Science, Seoul, Republic of Korea 2 Seoul National University College of Medicine, Department of Forensic Medicine, Seoul, Republic of Korea

Single nucleotide polymorphism (SNP) can be a valuable complementary marker to the conventional forensic STR marker in genetic typing, and its potential to provide advantages in the challenging forensic casework has been demonstrated. With the advent of high-throughput technologies, such as microarray and massively parallel sequencing, the usefulness of SNP typing is now expanding to a large-scale forensic application such as disaster victim identification. In this study, SNP analysis system on a microarray previously developed for forensic individual identification was applied to a real casework of forensic investigation in order to identify human body remains that had been remained unidentified by conventional forensic marker typing due to DNA degradation. DNA samples retrieved from bones were only able to be amplified for partial STR markers. Through SNP typing, 51 (15.6 %) remains of 327 the unidentified applied to this system were identified, which included the cases of relationship of 34 parent-child, 29 full-siblings, 14 uncle-nephew, and 10 grandparent-grandchild relationship with multiple reference family members. It was believed that higher recovery of genetic information in degraded samples and kinship analysis with multiple family members based on SNP data contributed to improved human remain identification. In addition, an automated kinship matching system along with the family reference database constructed for this work was helpful in large-scale kinship matching analysis as an efficient screening tool. This casework conducted by SNP analysis system demonstrated the effectiveness and efficiency of SNP typing for kinship analysis in forensic casework and a potential of a genetic typing system in large scale identification.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 171 P091 - IMPROVING THE DETECTION OF SEMEN ON NYLON FLOCKED SWABS AND FABRICS USING STANDARD PRESUMPTIVE TESTS Patrick Basset1, Christian Gehrig1, Frederic Grosjean1, Annalisa Grini1, Tacha Hicks1, Lydie Samie1, Vincent Castella1

1 Unité de Génétique Forensique, Centre universitaire romand de médecine légale - Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, Switzerland

The detection of spermatozoa and/or seminal fluid from sexual assault samples is essential to provide information regarding the biological source of the DNA, and thus help with issues that pertain to the activities alleged in the case. Within our laboratory, we use the Christmas tree staining (CTS) test in order to visualize spermatozoa, combined to the prostate specific antigen presumptive test (PSA) for the detection of the possible presence of seminal fluid. Until recently, we performed CTS on a few fibers that were taken from cotton swab or fabric. Because of this sampling procedure, the results were not necessarily representative of the whole stain, especially when the material was in very small quantities. This could lead to different results for the preliminary tests and the DNA analyses, which was problematic. In addition, from a practical point of view, it was difficult to retrieve fibers from flocked swabs as they are made of very short nylon fibers. Because of these issues, we designed a new protocol representative of the whole stain for both CTS and PSA. In this study, we compare the results obtained with the two protocols using three sperm dilutions (i.e. 1/50, 1/300 and 1/1200) deposited on cotton swabs (Prionics), nylon 4N6FLOQ swabs (Copan) and fabrics. Overall, the new protocol appears to be well suited to perform CTS and PSA on whole stains as well as efficient for recovering biological material from nylon swabs. Cutting the stains into small pieces resulted in improved sensitivity of CTS. In addition, the new protocol did not alter the quantity of DNA recovered with differential DNA extraction. These results led to the adoption of this new protocol into our laboratory procedures.

172 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P092 - IN-HOUSE VALIDATION OF FOUR COMMON PCR ASSAYS FOR AVIAN GENDER INVESTIGATION Thitika Kitpipit1, Wasananan Klungjan1, Nattakarn Kongmeka1, Supansa Bunpa1, Phuvadol Thanakiatkrai1

1 Prince of Songkla University, Applied Science, Hat Yai, Thailand

Gender identification in bird is important for both conservation program and forensic investigation. Since many bird species exhibit sexual monomorphism, this leads to many fraudulent cases in related communities. PCR-based techniques are the current standards for avian gender identification. Most assays target the specific introns of the chromo-helicase DNA binding gene (CHD) on sex chromosomes, which generate different fragment sizes on agarose gel electrophoresis. In this study, we validated four primer sets previously reported for avian sex determination (P2/ P8, CHD1F/CHD1R, 2550F/2718R, and 1237L/1272H) using 1,257 samples from 70 avian species commonly found in Thailand. The result showed that all four primer sets can be used for DNA analysis from avian species. However, the most efficient primer set vary depending on the species being investigated. Primer sets providing 100 % identification accuracy for each bird species are reported in this research.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 173 P093 - INTEGRATING MOLECULAR AND MORPHOLOGICAL DATA IN THE SECONDARY SEXUAL IDENTIFICATION OF MUSEUM SPECIMENS OF TAMANDUA TETRADACTYLA (XENARTHRA, PILOSA) Leonardo Cotts1, Ricardo Moratelli2, Dayse Silva3, Elizeu Fagundes Carvalho3, Silvia Martins4, Cesar Amaral3

1 Universidade Federal do Rio de Janeiro, Programa de Pós-graduação em Biodiversidade e Biologia Evolutiva, Rio de Janeiro, Brazil 2 Fundação Oswaldo Cruz, Fiocruz Mata Atlântica, Rio de Janeiro, Brazil 3 Universidade do Estado do Rio de Janeiro, Departamento de Ecologia - Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 4 Universidade do Estado do Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil

Xenarthra is a superorder of placental mammals constituted by extant armadillos, sloths, anteaters and their fossil relatives. Considering the biogeographic abundance, the two species included in theTamandua genus are the most expressive among the anteaters, being found of the north of Central America to the south of South America. Tamandua tetradactyla stand out as the most expressive species of anteater, with often records in brazilian biological collections. However, the absence of sexual dimorphism in the external morphology of T. tetradactyla and other anteaters contributes to that sexing mistakes are frequently observed in museum specimens. Here,we investigate the use of PCR amplification of the SRY gene (from SRY primers of human pY53.3 DNA sequence region; based in Takami et al.,1998)obtained from muscular tissues of T. tetradactylain the sexual identification of museum specimens. In addition, we used the molecular data to reveal possible intraspecific sexual variations in the skeleton of the analyzed samples. Twelve specimens of T. tetradactyla were sexed in this study from the SRY expression, being separated in nine males and three females.The results indicated clear misconceptions in the analyzed biological collections, since most of the specimens were previously identified as females. The anatomical data are, at the moment, concordant with the molecular analysis, with theappendicular bones of these specimenspresented the highest patterns of sexual distinctions in the examined skeleton.The molecular-morphological integration is probably a useful tool in the recognitionof intraspecific variation still unknown in museum specimens of Xenarthra. Keyword: Molecular sexing; SRY; Tamandua tetradactyla.

174 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P094 - INTERNAL VALIDATION OF GLOBALFILER™ KIT USING HALF VOLUME Eida Almohammed1, Fatma Al-Ali1, Hadi Sibte2

1 Ministry of Interior Qatar, Qatar Forensic Laboratory, Doha, Qatar 2 University of Central Lancashire, School of Forensic & Applied Sciences, Preston, United Kingdom

For approximately two decades, Microsatellites or Short Tandem Repeat (STR) markers have been the backbone for human identification testing in the forensic field. The expansion of STR multiplex kits can improve global sharing of STR profiles, rapid DNA typing, and DNA typing using high throughput sequencing. GlobalFilerTM Kit is highly sensitive and has extremely high power of discrimination. An internal validation of GlobalFiler™ Kit was carried out to test the robustness of this kit through a range of internal validation studies including half volume reaction, reproducibility, sensitivity, specificity, stability, mixture, analytical threshold, sensitivity & stochastic threshold, heterozygous balance & stutter threshold studies in accordance with SGWDAM guidelines. Reference and real casework samples previously tested using other autosomal STR kits from Qatar forensic lab were used during the validation. It was demonstrated that the GlobalFiler™ kit has enhanced discrimination power, is robust and extremely useful for forensic casework. Keywords: Globalfiler; Autosomal STR; Validation; Forensic; Qatar.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 175 P095 - INTERNAL VALIDATION STUDY OF THE NEXT GENERATION SEQUENCING OF GLOBALFILER™ PCR AMPLIFICATION KIT FOR THE ION TORRENT S5 SEQUENCER Christian Faccinetto1, Patrizia Serventi1, Nicola Staiti1, Alberto Marino1, Fabiano Gentile1

1 Reparto Carabinieri Investigazioni Scientifiche di Parma, Sezione Biologia, Parma, Italy

In the last few years, massively parallel sequencing (MPS) techniques have improved greatly in the field of forensic genetics, providing several advantages over the traditionally capillary electrophoresis (CE) system in terms of resolution, scalability, and throughput1,2. Nevertheless, before implementing MPS into routine forensic applications, both workflow and output results must be rigorously validated whit respect to robustness, performance and compatibility to CE-based data. We present the evaluation of MPS technology for short tandem repeat (STR) genotyping using Precision ID GlobalFilerTM NGS STR Panel v2 (Life Technologies) on the Ion S5™ platform (Life Technologies), including experiments to assess concordance, sensitivity, reliability and mixture analysis. MPS allowed to generate concordant result with those by CE system, demonstrating the reliability of both methods. Sensitivity study revealed that single source full-profile could be obtained using a significant less amount of DNA with compared to the quantity recommended in the protocol. In addition, STR genotypes obtained were reproducible and consistent among multiple typing replicates. Partial STR genotypes of minor contribute could be detected to 1:80 mixture. However, analysis of MPS-based STR revealed several noise artefacts (e.g. stutter artefacts) that were not independently filtered by Converge software NGS Analysis Module supported by Life technologies platform.

176 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P096 - INTERPRETATION OF SECONDARY BIOLOGICAL TRANSFER TRACES IN JUDICIAL GENETIC IDENTIFICATION Irina Streba1, Bulgaru Iliescu Diana2, Capilnean Alin2

1 Institute of Legal Medicine Iasi, Forensic Identification, iasi, Romania 2 Institute of Legal Medicine Iasi, Forensic Pathology, Iasi, Romania

The interpretation of biological transfer traces is a challenge that requires on the one hand as much data on crime scene investigation and on the other hand pretests that show the nature of the trace that was genotyped. The transfer of biological traces can be primary (direct) and secondary (indirect). In the case of primary transfer the victim’s, perpetrator’s or witness’s biological product is deposited directly on the body of another person or on divers objects. Secondary transfer occurs when the biological trace is transferred to the victim, suspect or an object by an intermediate vector. In this case the transfer is indirect and there is no direct physical contact between the source and support. The vector can be a person or object. We will present a case of secondary transfer encountered in the practice of our Laboratory and the interpretation of the presence of these biological traces in the context of the case. Taking into account the victim’s investigation data and statements, most of the contact traces were performed by primary transfer and justified by the relationship between the victim and the aggressor. The traces of a single object of clothing belonging to the aggressor could have been produced by secondary transfer and their existence criminalizes them. Keywords: biological traces, secondary transfer, identification.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 177 P097 - INTERSEXUALITY AS A POTENTIAL SOURCE OF ERROR IN SEX DETERMINATION USING FORENSIC MULTIPLEX KITS Bettina Dunkelmann1, Katharina Helm1, Ines Grießner1, Waltraud Zahrer1, Müller Eva1, Tamara Kastinger1, Gabriele Kreindl1, Jan Cemper-Kiesslich1, Franz Neuhuber1

1 University of Salzburg, Institute of forensic science, Salzburg, Austria

The gender specific marker amelogenin is a valuable tool in sex determination. In all commercial kits used in forensic genetics the amelogenin locus is used as a marker for gender determination. However, gonosomale aberrations, deletions or primer binding site mutations can lead to incorrect genotyping. Therefore, misleading results can be obtained due to the phenotype or genetic intersexuality even though the genotyping is correct. Thus, gender-determining test results must always be critically questioned to avoid misguided criminal investigations. In our routine casework we observed six cases with discrepancies regarding the biological and the „legal“ gender. Further validation revealed in three cases a deletion on the Y-chromosome encompassing the AMELY and other Y-STR markers on the short arm of the Y-chromosome. In another case we observed a person with phenotypically male characteristics with two X-chromosomes and no genetic indication of a Y-chromosome. In the last two cases the examined individuals show phenotypically female characteristics but do have a XY-constellation in the gender specific marker amelogenin. This shows that there can be numerous variants with divergent biological backgrounds. Similar findings are well known in the forensic society, but they are seldom and can be complex. Consequently these potentially misleading genetic anomalies should always be investigated and published if detected. Furthermore, carefully worded statements in reports and in court trials should be standard.

178 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P098 - INTRA - BONE NUCLEAR DNA VARIABILITY IN SECOND WORLD WAR METATARSAL AND METACARPAL BONES Irena Zupanič Pajnič1, Jezerka Inkret1, Barbara Gornjak Pogorelc1, Gregor Haring1, Tomaž Zupanc1, Jože Balažic1, Eva Podovšovnik2

1 University of Ljubljana, Medical Faculty, Institute of Forensic Medicine, Ljubljana, Slovenia 2 University of Primorska, Turistica, Faculty of Tourism Studies, Portorož, Slovenia

DNA analysis of WWII skeletal remains are challenging because of limited yield of DNA that is usually recovered from poorly preserved old bones. Recent forensic research has focused on determining which skeletal elements are superior in their preservation of DNA and little focus has been placed on measuring intra-element variation. Since it is imperative to direct sampling efforts at bone sections that will yield maximal recovery of genetic material, the goal of our study was to explore intra-bone variation in DNA content by measuring nuclear DNA quantity and quality in metatarsal and metacarpal bones. To exclude the influence of taphonomic issues (post-mortem interval and the environment to which the remains were subjected) we examined 213 bones from a single WWII mass grave to ensure that skeletons were decomposed for the same duration and under the same conditions. From each bone DNA was extracted from compact diaphysis (extraction 1) and from spongy epiphyses (extraction 2). Half gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen) device. DNA content and rates of DNA degradation in the different bone tissues of metatarsal and metacarpal bones were determined with the PowerQuant kit (Promega). Up to 13.4 ng DNA/g of powder was obtained from metatarsal diaphysis and up to 62 from metatarsal epiphyses. Higher difference was found in metacarpals where up to 18 ng DNA/g of powder was obtained from diaphysis and up to 240 from epiphyses. On average, twelve times more DNA was obtained from metatarsal epiphyses than diaphysis, and 26 times more from metacarpal epiphyses than diaphysis. We found out that the best place to sample from within metatarsal and metacarpal bones are spongy epiphyses.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 179 P099 - INVESTIGATION INTO THE TRANSFER OF MICROBIOMES WITHIN A FORENSIC LABORATORY SETTING Ana Neckovic1, Roland van Oorschot2, Bianca Szkuta1,2, Annalisa Durdle1

1 Deakin University, School of Life and Environmental Sciences, Waurn Ponds, Australia 2 Victoria Police Forensic Services Centre, Office of the Chief Forensic Scientist, Macleod, Australia

Microbial profiling within forensic science is an emerging field that may have applications in the identification of individuals using microbial signatures. It is important to determine if microbial transfer may occur within a forensic laboratory setting using current standard operating procedures (SOPs) for DNA recovery, to assess their suitability for microbial profiling and establish potential limitations. This study investigated the presence and potential transfer of human microbiomes within a forensic laboratory. Swabs of laboratory surfaces, personal pro- tective equipment (PPE) and equipment were taken before and after a mock examination of a cotton swatch harbouring microbiota transferred from direct hand-contact. Comparative analyses of the microbial profiles obtained from the laboratory surfaces, external surfaces of the PPE worn by the participant and the cotton swatch were performed to determine potential source contributions. Reference microbial profiles of the researcher and participant were included for comparative purposes to the experimental microbial profiles. Results reveal a potential transfer of microbiota between the examined material, participant and/or researcher, and laboratory equipment and surfaces, highlighting potential issues regarding microbial profiling using current laboratory SOPs. These issues include the potential presence of contaminating or competing microbial DNA from human-associated microbiomes or laboratory settings, which may interfere with forensic samples considered to have low-biomass or microbial communities of interest. Therefore, current DNA Recovery SOPs may not be suitable for work with human microbiomes in a forensic laboratory setting.

180 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P100 - INVESTIGATION OF PARENTAGE OF MENDEL GRAPEVINE Rosa Arroyo-Garcia1, David Carrasco1, Nami Goto-Yamamoto2, Jiří Drábek3

1 CBGP-INIA- Campus de Montegancedo- Autovía M40 km 38- Pozuelo de Alarcón- 28223, Dpto. Biotecnología, Madrid, Spain 2 National Research Institute of Brewing, n.a., Higashi-Hiroshima, Japan 3 Palacky University, IMTM- Faculty of Medicine and Dentistry, Olomouc, Czech Republic

Johann Gregor Mendel is recognized as one of fathers of genetics. His hybridization experiments were done not only with pea but also with other plants, including Vitis spp.(grapevine). Due to its vegetative way of propagation, Mendel grapevine with faintly purple grapes and serrated leaves with backside surface covered with fine hairs is still in existence. By quirks of the destiny, its scion was sent to Tokyo, Japan by Siberian Railway in 1914 and its twig was sent back to Brno (then Czechoslovakia, now the Czech Republic) in 1989. Now, there are four vines in Mendelia- num in Brno and two vines in Koishikawa botanical garden of the University of Tokyo. However, the grapevine parents and/or variety are not known because Mendel´s monastery successor Rambousek burnt almost all the scientific protocols. By DNA profiling of 20 microsatellite loci (VMC1b11, VVIH54, VVIN73, VVIB01, VVMD7, VVIQ52, VVMD24, VVIP60, VVIN16, VVMD5, VVIV37, VVMD28, VVMD27, VVIV67, VMC4f3, VVIp31, VVMD21, VVMD25, VVMD32, and VVS2), using grapevine STR (SSR) databases available online or as manuscript supplements, and kinship software, we attempted to reveal Mendel grapevine parents or kins. Here we present our preliminary data together with a discussion of differences in quality parameters of human and Vitis STR databases. Acknowledgements: Supported by RTA2014–00016–03–01, CZ.02.1.01/0.0/0.0/16_013/000167 4, IGA_LF_2019_003, and NPU LO1304.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 181 P101 - IS IT RELIABLE TO USE PARAFFIN EMBEDDED HISTOLOGICAL TISSUES AS BIOLOGICAL EVIDENCE IN PARENTAGE CASES? Ayse Serin1, Husniye Canan1

1 University of Cukurova, Forensic Medicine, Adana, Turkey

Short tandem repeats (STR) are reliable markers used for forensic identification and paternity testing. In some cases, histological paraffin blocks containing malignant cells can be used for STR typing in identification and paternity. DNA profiling and result interpretation with these samples often becomes a challenging job. The malignant tissues may show preferential amplification, loss of heterozygosity, and microsatellite instability. Due to the suspected paternity case, two paraffin blocks were sent for DNA analysis by Court of Justice. Pathology report of the paraffin blocks have shown that they contain lung cancer tissues taken with bronchoscopy. After extracting DNA with silica based method from the samples taken from different parts of these two blocks, 21 autosomal STR loci and 17 gonosomal STR loci were studied. When we have compared the STR profiles, Y-chromosomal STR loci were matched fully. But 2 of the 21 autosomal STR loci were found mismatches in father-son comparing. These two mis- matched loci have homozygosity for the father. Moreover, 10 of 21 autosomal loci have shown homozygosity, and at one locus D16S539 has an additional allele. While increased homozygosity has increased the paternity index, mismatches loci have lessened unlike the actual value. These results suggest that great care should be taken in the evaluation of the DNA typing results obtained from clinical cancerous specimens, in particular when no other reference samples containing normal tissue are available.

182 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P102 - ISOLATION OF PURE SPERM CELLS FROM SEXUAL ASSAULT MOCK ANAL SWABS USING DEPArray™ DIGITAL SORTER Zhen Xu1, Tzuming Wang2, Zheng Tu1, Yongjiu Lee1

1 Institute of Forensic Science- MPS- China, Division of Forensic Genetics, Beijing, China 2 Biocommander International Co.- LTD., Research & Development, Taipei, China

Anal swabs are commonly collected as part of Sexual Assault Kits (SAKs) in forensic rape investigations. Standard analysis of such samples, differential DNA extraction and genotyping is often hampered by the unbalanced presence of complaint versus suspect DNA as well as PCR inhibition from bacteria and feces. DEPArray™ technology (Menarini Silicon Biosystems, MSB) has extensively been reported to enable the separation of pure pools or single cells from biological mixtures. Up today, in the context of sexual assault investigation, this technology has been applied only to vaginal swabs or garments: here we describe for the first time the possible application to anal swabs. To simulate SAKs samples, 600 ul of fresh semen obtained from a volunteer male donor was injected into a female volunteer’s anus, two anal swabs were collected 1 hour after injection, one of which showed clear feces residues. Samples were prepared using DEPArray™ Forensic SamplePrep Kit and analyzed with DEPArray™ NxT. Sperm Cells and Epithelial Cells were identified and recovered in pools from both swab samples. Isolated cells were lysed with DEPArray™ LysePrep Kit and amplified using AmpFℓSTR® NGM SElect™ PCR amplification kit (ThermoFish- er Scientific), according to manufacturer’s instructions. Capillary Electrophoresis has been performed on a 3500 Genetic Analyzer (Applied Biosystems). A single-source, donor-matching, almost complete (83 % of the expected alleles) male profile was obtained from as little as 16 Sperm Cells. Epithelial cells recoveries provided as well matching female profiles. This preliminary work promises that multiple types of biological mixtures may be resolved by DEPArray™ technology to obtain pure or even single cells, overcoming DNA mixtures interpretation issues.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 183 P103 - JANE DOE’S IDENTIFICATION FROM TEETH BY AUTOMATE DNA EXTRACTION Raluca Dumache1, Enache Alexandra1

1 Victor Babes University of Medicine and Pharmacy Timisoara- Romania, Forensic Medicine- Bioethics- Deontology and Medical Law, Timisoara, Romania

Identification of humans from their skeleton remains by DNA genotyping represents an important side in forensic genetics. We present the identification of an unknown person from the skeleton remains by automated DNA technology. As DNA reference sample from Jane Doe, we used the right incisor. After washing and cleaning of the teeth, the pulverization was performed by MillMix 20 (Domel, Slovenia). The DNA extraction was done on an automate Maxwell16 FSC (Promega, USA) using the DNA IQ Casework Pro Kit (Promega, USA). Further, the PowerQuant System (Promega, USA) was used for the DNA quantification of the sample. The quantification was performed on an ABI 7500 (ThermoFischer, USA) using SDS Software 2.0.6. The extracted DNA was further amplified on a ProFlex PCR System ( ThermoFischer, USA) using the AmpFL- STR Identifiler Plus Kit (ThermoFischer, USA). All DNA samples were amplified in duplicate. In all amplification reactions a positive and a negative PCR control were used. PCR products were further run on a 3500 Genetic Analyzer (ThermoFischer, USA). Data analysis was performed by GeneMapper 1.4 (ThermoFischer, USA). For the confirmation of Jane Doe’s identity, the obtained DNA profile was compared to her mothers’ DNA profile and it was a match. In the process of DNA extraction from teeth, bones or old human remains to reduce handling errors and increase the choice of obtaining good quality DNA, the automate platform is a good option to use instead of the manual procedure.

184 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P104 - KINSHIP ANALYSIS ON SKELETAL ANCIENT REMAINS: THE CASE OF “EL CERRO DE LA HORRA” (BURGOS, SPAIN) Sara Palomo-Díez1,2, Carlos Baeza-Richer1,2, Ángel Esparza-Arroyo3, Javier Velásco-Vázquez4, Cláudia Gomes1,2, Alejándra Sánchez-Polo3, Ana María López-Parra1,2, Antonio Blanco-González3, Eduardo Arroyo-Pardo1,2

The finding of a multiple simultaneous burial, usually, entails the consideration of a close biological relationship among the individuals. In this context, this paper focuses on three very closely interred sub-adults in the site of El Cerro (La Horra, Burgos, Spain), a pit site dated on the Middle Bronze Age of the Central Iberian Plateau, ca. 1850–1450 cal BC. In the simultaneous triple burial two of the subadults inhumed were placed in lateral decubitus position intertwined face to face: a 7–8 years-old infant placed on his/her right side (HOR 02) and a second infant, perhaps male, about 11 years old, on the left (HOR 03), keeping a purpose- fully symmetrical disposition. On top of these corpses, and occupying the space between them, was placed a third subadult (HOR 01), a juvenile of 11–15 years, probably female. We have selected different teeth samples from each one of these individuals for genetic analysis, with the aim of enlightening their alleged kinship relationship, which could be key in the interpretation of the site and the overall cultural practices. To perform the kinship analysis, we have extracted and amplified DNA from three dental pieces from individuals HOR01 and HOR02, and two from HOR03. The DNA markers analysed were: autosomal short tandem repeats (STRs), autosomal insertion – delection markers (InDels) and HVI and HVII regions of mitochondrial DNA (mtDNA). Nevertheless, the poor preservation state of the DNA did not allow the obtention of reliable results autosomal markers. However, the mitochondrial DNA analysis provide the same haplotype to the three individuals (16298C 64T 72C, in the range: (16105–16399) (57–128)), belonging to HV0 mitochondrial haplogroup. The results point to a kinship among all the individuals through the maternal lineage.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 185 P105 - ‘KITOME’: THE KRYPTONITE OF FORENSIC METAGENOMICS Gabriella Mason-Buck1, David Ballard1, Rebecca Smith1, Ye Jee Roh1, Denise Syndercombe Court1

1 King’s Forensics, King’s College London, London, United Kingdom

Progress in forensic genomics has enabled the exploitation of trace material beyond previous limitations. When the identification of a perpetrator is paramount to an investigation, this can be achieved with a reference profile or a hit to a National DNA database. If this proves unsuccessful, investigative intelligence tools such as inference of ancestry and hair and eye colour prediction can be deployed. Such tools provide extra information about the perpetrator, leading to the suspect pool being reduced and prevents the case turning cold. Metagenomic studies can explore all genomic material present within an environment: bacterial, fungal, viral, human, plant and animal DNA. From a forensic perspective, the metagenomic profile left by or present on the hands of a perpetrator can offer intelligence as to where that person may have been and what they have been in contact with before, during and after a crime event. This method moves away from targeted sequencing of specific regions (such as 16S rRNA for bacteria) and towards simultaneous sequencing of trace level taxonomic material from non-human DNA. Sequence data obtained using Massively Parallel Sequencing (MPS) can be assigned via bioinformatics pipelines that search against multiple taxonomic databases. An important consideration with this type of analysis is the background taxonomic profile within the reagents and consumables used. This can be referred to as the ‘Kitome’. Data presented here will highlight the species that have been consistently identified as contaminating taxa, their potential origin within the analysis process and the consequence of this for accurate analysis.

186 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P106 - MASSIVE PARALLEL SEQUENCING AND OSTEOGENESIS IMPERFECTA: AN ESSENTIAL TOOL FOR FORENSIC INVESTIGATION OVER CHILD ABUSE Valerio Onofri1, Mauro Pesaresi1, Chiara Turchi1, Adriano Tagliabracci1

1 Polytechnic University of Marche-Ospedali Riuniti, Department of Biomedical Sciences and Public Health- Section of Legal Medicine, Ancona, Italy

Osteogenesis imperfecta (OI) is a rare disease of collagen synthesis causing bone fragility. Also called “glass bone disease” since it manifests as spontaneous fractures, it is classified into nine types, both dominant and recessive transmission. In 95 % of cases OI is caused by mutations in COL1A1 and COL1A2 genes encoding the alpha1 and alpha2 chains of type 1 collagen, mainly null variants caused by frame-shift/nonsense mutations or splicing defects. In infants the differential diagnosis include not-accidental trauma, so child abuse. Families suspected of abuse often provide an unverified history of frequent fractures; conversely, the family history of individuals with OI often does not reveal any other affected individuals because of a de novo pathogenic variant in the proband or the presence of a mild phenotype in relatives. Therefore, legal medicine unit with DNA lab is crucial in these cases since it could early collect living or autopsy samples when a child abuse is suspected and then test DNA. We set up a MPS panel including the coding regions of COL1A1 and COL1A2 and other 11 genes known to cause OI. Six cases were studied, two of them consisting of suspected abuses in two living child. MPS libraries were sequenced by Ion Torrent PGM platform; pathogenic variants and VUS (variants of uncertain significance) were confirmed by Sanger sequencing and familial segregation study was performed in three cases to better characterize the clinical significance of the mutation. This study remarks that MPS could help not only for identification, ancestry/phenotyping or sudden cardiac death applications but also for forensic investigation over child abuse. The usefulness of this assay for diagnostic projects on victims of abuse together with post-mortem cases is discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 187 P107 - MASSIVELY PARALLEL SEQUENCING OF AUTOSOMAL STRs AND IDENTITY-INFORMATIVE SNPs WITH THE FORENSEQ SYSTEM HIGHLIGHT CONSANGUINITY IN SAUDI ARABIA Yahya Khubrani1,2, Mark A. Jobling1, Jon Wetton1, Pille Hallast3

1 University of Leicester, Department of Genetics & Genome Biology, Leicester, United Kingdom 2 General Administration of Criminal Evidence, Forensic Genetics Laboratory, Riyadh, Saudi Arabia 3 Wellcome Trust Sanger Institute, Department of Genetics, Cambridge, United Kingdom

Massively parallel sequencing (MPS) reveals more diversity than capillary electrophoresis including different combinations of diverged repeat units, along with indels and SNPs in both the repeat array and flanking regions. MPS can also simultaneously analyse many loci in a single test, simplifying analysis of different marker types and making best use of limited casework material. MPS allows amplicon size to be minimized, ideal for the Middle East where high average temperatures favour short-amplicon STRs and iiSNPs that provide high discrimination from degraded DNA. The ForenSeq™ DNA Signature Prep Kit allows the simultaneous amplification, with one primer mix, of over 150 loci including autosomal and Y-STRs plus X-STRs and identity informative SNPs (iiSNPs). DNA profiles of 89 Saudi males generated with ForenSeq primer mix A gave an average depth of coverage per individual of 15,656 reads for STRs and 465 for iiSNPs. Among the 4,802 autosomal STR alleles, there were 238 length variants and 341 repeat sequence sub-variants across 27 loci, including several not in the STRait Razor v3 database. Also, among the 91 iiSNP loci, 26 had additional variants within the regions flanking the target SNP, further increasing discrimination power. Most loci display an excess of observed homozygosity reflecting the high frequency of endogamous cousin marriages in Saudi Arabia, combined with population structure at both autosomal and Y-STRs which leads to clusters of individuals that share a common heritage. This reduces the discrimination of the multiplexes relative to more diverse and exogamous societies, as shown by comparisons with the same loci in the CEPH-Human Genome Diversity Panel (HGDP).

188 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P108 - METAPOPULATION ANALYSIS OF THE BACTERIAL 16S RNA GENE AS A TOOL FOR IDENTIFYING THE PREPUTIAL SPECIMEN OF A MAN Jolanta Powierska-Czarny1, Paweł Cyplik2, Joanna Idkowiak1, Monika Antczak1, Łukasz Kaczorowski1, Bartosz Hornik1, Aleksandra Górka1, Agnieszka Piotrowska-Cyplik3, Jakub Czarny1, Magdalena Chrostowska1

Only biological material such as blood, semen, saliva and urine can be effectively and simply identified in routine forensic examinations based on simple and cheap enzymatic and immu- nochemical tests. In some cases of sexual crimes, when the ejaculation of semen does not occur and the genetic profile of the suspect is identified in the evidence samples, it is necessary to indicate whether the revealed biological material originates from a prepuce to press effective charges. Analysis of 27 preputial swabs of males suspected of sexual crimes in which no other genetic material was found (sexual partners). The DNA was isolated using the QIAsymphony DNA Inves- tigator Kit (Qiagen) and a QIASymphony workstation. Regions 2, 3, 4, 5, 6, 7, 8 and 9 of the 16S RNA gene were amplified using the Ion 16S™ Metagenomics Kit (Life Technologies). Libraries were prepared using the Ion Plus Fragment Library Kit (Life Technologies) and library amplification was carried out based on the Ion PGM™ Hi-Q™ View OT2 Kit using the Ion OneTouch™ 2 System (Life Technologies). Sequencing was carried out using an Ion Personal Genome Ma- chine™ (PGM™) System (Life Technologies) with the Ion PGM ™ Kit Hi-Q™ View Sequencing Kit (Life Technologies) and Ion 316™ Chip Kit v2 BC (Life Technologies). The bacterial microflora under the prepuce is characterized by a different composition compared to that found in other environments. Based on the test results, a group of bacteria characteristic for the studied environment can be selected. The obtained results indicate that studies regarding the composition of bacterial microflora may be a useful tool for forensic identification of preputial specimen.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 189 P109 - MITOCHONDRIAL DNA ANALYSIS IN THE UNITED ARAB EMIRATES POPULATIONS Reem Almheiri1, Rashed Alghafri1, Mayda Tracy Bakri1, William Goodwin2

1 Dubai Police G.H.Q, General Department of Forensic Science and Criminology, Dubai, United Arab Emirates 2 University Of Central Lancashire, School of Forensic and Applied Sciences, Manchester, United Kingdom

Mitochondrial DNA analysis has earned its place in the field of genetics for providing a new window into understanding population ancestry. Its ability to produce results from minimal or decayed samples where nucleus DNA profiling proves difficult is an additional benefit to forensic genetics. As a result, it is essential to begin building a more precise regional study of populations. In this study, 530 whole blood samples were collected on FTA paper, from the United Arab Emirates populations, including inhabitants from rural regions. The three main ethnic groups studied were Indians, Pakistanis, and Emiratis. Both extraction of whole blood samples using PrepFiler®as well as direct amplification of mtDNA control region from purified 1.2 mm disc of FTA card stained with blood were attempted in this study. Quantity of the amplified control region was estimated using gel electrophoresis. Three sets of primers were used afterward to sequence purified products of amplified control region of mtDNA using Sanger sequencing method. Whole mtDNA sequence will also be presented for same set of samples, generated using MPS technology – Ion™ 5S for concordance purposes. Resulted haplotypes were compared against worldwide mtDNA database (EMPOP) and estimated haplotypes frequency is shown in this study. As a forensic lab, mock samples were also tested to highlight the potentials value of using mtDNA analysis for both methods. This study helps extending the current available mtD- NA control region database and also investigate the whole mtDNA sequence polymorphisms of some of these samples. As a result this study shows great value of implement MPS in the routine work in forensic genetics at Dubai Police.

190 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P110 - MITOGENOME SEQUENCE VARIATION IN HAIR SHAFTS: A PILOT-STUDY Vania Pereira1, Gabriela Huber2, Christina Stobl2, Walther Parson2,3

1 Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 2 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 3 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

The application of mtDNA in forensics genetics has proven to be an important tool in the analysis of trace samples where nuclear DNA is degraded and/or present in low amounts. The generation of an mtDNA profile from standard reference samples is fairly simple with the current sequencing technologies. However, forensic casework samples still pose challenges in the interpretation and reporting of mtDNA evidence, especially in regards to heteroplasmy. This work focuses on mtDNA sequence variation in hair shafts. It is known that hair shafts show a tight bottleneck in the segregation of mtDNA molecules during hair development. This can result in heteroplasmy along the hair shaft, or even lead to fixation of a variant that differs from the haplotype observed in other tissues. Although studies have addressed this issue for the mtDNA control region, heteroplasmic levels in the mtDNA coding region in hair are generally unknown1-4. In this study, we investigated mtDNA heteroplasmy along hair samples. Hair shafts were further divided into fragments that were extracted and sequenced independently using massively parallel sequencing (MPS). The heteroplasmic levels detected along and among hair shafts were compared to those reported for blood and buccal reference samples. This is a pilot-study with few samples, and more data are needed to address how to report heteroplasmy in different tissues, and define thresholds for data generated with different MPS platforms. We invite all interested researchers to contact us and collaborate on this project. References 1. Tully, G. et al. 2004. Forensic Sci Int 140(1):1–11 2. Paneto, G.G. et al. 2007. Forensic Sci Int 173(2–3):117–121 3. Paneto, G.G. et al. 2010. J Forensic Sci 55(3):715–718 4. Roberts, K.A. et al. 2011. J Forensic Sci 56(1):46–60

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 191 P111 - MOLECULAR DISSECTION OF A CRIME SCENE – INTRODUCING STR SEQUENCING IN ROUTINE INVESTIGATION Marta Diepenbroek1, Birgit Bayer1, Katja Anslinger1

1 Ludwig Maximilian University of Munich, Institute of Legal Medicine, Munich, Germany

Progress is an integral part of science and therefore it is no surprise that also forensics is currently undergoing a change. With Massively Parallel Sequencing we are able to perform a molecular dissection of DNA in order to shed more light onto a criminal investigation. However, the new insights brought by forensic genomics, bring new challenges concerning the data analysis and interpretation. In the past few years, due to a significant number of validation studies, MPS has proven its potential to change our gold standards of STR analysis. Some of the issues still stop- ping MPS from becoming a standard tool among the forensic community will most likely solve themselves with time (e.g. nomenclature standards or national databases) but others can be resolved only with learning-by-doing. And for this to happen more laboratories have to decide on introducing MPS into their routine work so that its impact on crime scene investigation can be presented to a broader range of the most crucial partners: the law enforcement. In this study we investigated 57 challenging samples which represented the cross-section of the caseworks we face routinely in our institute. The samples were amplified and sequenced in duplicate using Precision ID GlobalFiler™ NGS STR Panel v2 and Ion GeneStudio S5 (ThermoFish- er Scientific). For data analysis we used the Converge™ Software v2.1. The samples selected for the study were classified into 3 groups: diverse biological material (bones, teeth and muscles), current caseworks (crime scene traces and suspects samples) and cold cases (low DNA quality and quantity). Here, we present the difficulties while analyzing the most challenging forensic samples but also we demonstrate the power of the results given by MPS when compared to the standard CE outcome.

192 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P112 - MOLECULAR IDENTIFICATION AND GENETIC DIVERGENCE OF NEW-WORLD CALLITHRICHINAE MARMOSETS BASED ON THE MITOCHONDRIAL COI GENE Cesar Amaral1, Elizeu Fagundes Carvalho1, Silvia Martins2, Dayse Silva1

1 Universidade do Estado do Rio de Janeiro, Departamento de Ecologia - Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 2 Universidade do Estado do Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil

The New World marmosets of the sub-family Callitrichinae includes genera of marmosets, tamarins, and lion tamarins. They are small, arboreal, diurnal, insectivore/frugivores inhabiting forest- ed, rural, and urban areas of tropical Central and South America. About 23 species are currently recognized. The marmosets are currently divided into four genera, the Callithrix, Cebuella, Cal- libella, and Mico with Callimico being regarded as a sister species to all these marmosets. With several threatened species, marmosets conservation are constantly under debate. Several management plans and actions are now in course although a precise delimitation of the operational management units are still open. Here we analyzed and discussed the molecular identification and the COI-K2P genetic divergence patterns among these marmosets based on newly determined and previously published sequences under a DNA barcoding perspective. The use of DNA barcoding methodology, which uses a short mitochondrial DNA region of the COI gene is one of the most used approaches for biodiversity assessment and species molecular identification. Patterns of COI divergence could be helpful on a more precise delimitation of the operational taxonomical units that could provide knowledge for a more comprehensive approach on the conservations of these species. Keywords: Callithrix; barcoding; marmosets.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 193 P113 - MOLECULAR IDENTIFICATION OF POISONOUS PUFFERFISHES AND CROSS-ATLANTIC GENETIC DIVERGENCE PATTERNS BASED ON THE MITOCHONDRIAL COI GENE Rodrigo Gondenberg-Barbosa1, Dayse Silva2, Elizeu Fagundes Carvalho2, Silvia Martins3, Cesar Amaral2

1 Universidade Federal do Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 2 Universidade do Estado do Rio de Janeiro, Departamento de Ecologia - Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 3 Universidade do Estado do Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil

The Tetraodontidae is the most speciose family within the order Tetraodontiformes, being characterized by beak-like jaws and the presence of powerful neurotoxins Tetrodotoxin/Saxi- toxin associated with soft tissues, inflation behavior under stress, a condition shared with its accepted sister-family Diodontidae. Although several studies, including both morphological and molecular analyses,have been conducted in the last decade the phylogeny and biogeogra- phy of Tetraodontidae and its species remain under debate. Several fatal intoxication cases had been observed in the last years related with the ingestion of Tetraodontidae species all around the world. Although recent technological advances have facilitated the sequencing of an entire mitogenome (~16Kb), increasing the use of mtDNA as a phylogenetic marker, several studies primarily focusing on small mtDNA regions are continuously conducted. Therefore, the aim of the present study was to investigate the molecular identification and the cross-atlantic genetic divergence patterns observed from the mitochondrial COI gene of the poisonous genus Spho- eroides based on newly determined and previously published sequences from both North and South Atlantic Oceans. Keywords: Sphoeroides; mitochondria; COI; pufferfishes.

194 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P114 - mRNA MPS TISSUE IDENTIFICATION ASSAY TO AID IN THE INVESTIGATION OF TRAUMATIC INJURIES Erin Hanson1, Andrea Salzmann2, Guro Dorum2, Cordula Haas2, John Ballantyne1,3

1 University of Central Florida, National Center for Forensic Science, Orlando, USA 2 University of Zurich, Zurich Institute of Forensic Medicine, Zurich, Switzerland 3 University of Central Florida, Department of Chemistry, Orlando, USA

Molecular analysis of the RNA transcriptome from a putative tissue fragment should permit assignment to a specific organ since each tissue will exhibit a unique pattern of gene expression. Determination of the organ source of tissues from crime scenes may aid in shooting and stabbing investigations. We have developed a new prototype massively parallel sequencing (MPS) mRNA profiling assay for organ tissue identification, designed to definitively identify 13 organ/ tissue types using a targeted panel of 48 mRNA biomarkers. The identifiable organs and tissues include brain, spinal cord, lung, trachea, liver, skeletal muscle, heart, kidney, adipose, intestine, stomach, skin and spleen. The biomarkers were chosen after iterative specificity testing of numerous candidate genes in various tissue types. The assay is very specific with little cross reactivity with non-targeted tissue, and can detect RNA mixtures from different tissues, including two- to five-tissue admixtures.The sensitivity of the assay was evaluated as well as assay reproducibility between library preparations and sequencing runs. We also demonstrate the ability of the assay to successfully identify the tissue source of origin in cadaver samples, tissue samples with varying post mortem intervals (PMI) and mock and bona fidecasework samples. We used the data to train a multivariate statistical model that predicts the tissue type based on the mRNA profile. By considering co-expression of markers the model can recognize distinct expression patterns in each tissue.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 195 P115 - MULTIPLEX DNA METHYLATION PROFILING BY ARMS‑PCR FOR BODY FLUID IDENTIFICATION Huan Tian1, Yu Tan1, Zhilong Li1, Duo Peng1, Weibo Liang1, Lin Zhang1, Dan Chen2, Tao Feng2, Peng Bai1

1 Sichuan University, Department of Forensic Genetics- West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Chengdu Public Security Bureau, Department of Forensic Genetics, Institute of Forensic Science, Chengdu, China

Identifying the origin of body fluid samples that recovered at crime scenes could provide important information for the reconstruction of the crime scenes. In this research, a set of body fluid-specific CpG sites, USP49, cg26079753–7d, cg08792630 and cg16732616, were developed to specifically detect the patterns of DNA methylation using the Amplification refractory mutation system-PCR (ARMS-PCR) in order to identify semen, vaginal fluid, blood and saliva samples respectively. DNA of human samples were bisulfite modified using commercial bisulfite treatment kit. ARMS-specific primers were used to amplify the CpG sites and the methylation values of each CpG site were detected by capillary electrophoresis. Finally, the methylation profiling of body fluid samples could be determined by multiplex ARMS-PCR assay. We proved that this method was robust and effective to identify body fluids. It is the first time to apply the ARMS- PCR to detect DNA methylation in forensic science, which provides a new idea for forensic body fluid identification.

196 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P116 - MUTATION RATES OF SIX Y-CHROMOSOMAL STR LOCI ESTIMATED FROM 210 PEDIGREES IN CHINESE HAN POPULATION Yiping Hou1, Jing Liu1, Hai Liu2, Zheng Wang1, Xianhua Qiao2, Hong Zhu1

1 Sichuan University, Institute of Forensic Medicine- West China School of Basic Science & Forensic Medicine, Chengdu, China 2 Henan Provincial Public Security Bureau, The Institute of Forensic Science and Technology, Zhengzhou, China

The Y-chromosomal short tandem repeats (Y-STRs) with low mutation rates as slowly mutating (SM) Y-STRs could be used to refine the construction of phylogenetic trees in evolutionary research. Six Y-STR markers (DYS388, DYS426, DYS461, DYS485, DYS525 and DYS561) were from the previously published paper and considered as SM Y-STRs. Here, we investigated the six Y-STRs from 210 pedigrees in Chinese Han population in Henan province, covering an overall number of 8178 meiotic transfers and the number of meiosis of one pedigree varied from 1 to 15. The number of mutations of the six Y-STRs ranged from 0 (DYS388 and DYS426) to 12 (DYS561), resulting the observed mutation rates spanned from 0 (95 % credible interval [CI], 0 ~ 2.196 × 10-3) to 8.804 × 10-3 (95 % CI, 4.557 × 10-3 ~ 1.533 × 10-2). According to the results, only DYS388 and DYS426 could be classified as SM Y-STRs in the Chinese Han population. Although these results will replenish the data of Y-STR mutations as well as be important for paternal genealogy identification, kinship analysis, and phylogenetic tree reconstruction, the mutation rates of the six Y-STRs were significantly different among different populations, which we need to pay attention to in future studies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 197 P117 - MVC: AN INTEGRATED MITOCHONDRIAL VARIANT CALLER FOR FORENSICS Chantal Roth1, Walther Parson2, Christina Strobl2, Robert Lagacé1, Marc Short1

1 Thermo Fisher Scientific, Human Identification, South San Francisco, USA 2 Medizinischen Universität Innsbruck, Institut für Gerichtliche Medizin, Innsbruck, Austria

Mitochondrial DNA (mtDNA) sequencing is a valuable forensics tool in determining the source of DNA obtained from degraded, damaged or small biological samples. However, despite the recent advances in Massively Parallel Sequencing (MPS), the analysis of mtDNA has remained challenging: alignments can be misleading and may result in incorrect variant calls, and contamination from nuclear DNA from small samples may obscure true variants. In this poster, we discuss an integrated approach to solving these issues in a new mito variant caller that inte- grates various sources of knowledge about mtDNA, including phylotree, EMPOP and NUMTs statistics, that avoids many of the pitfalls of standard algorithms. The knowledge is integrated into the alignment algorithm itself to distinguish true variant calls from NUMT contamination and sequencing artefacts.

198 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P118 - MYOCARDIAL TRANSCRIPTOME ANALYSIS IN SUDDEN UNEXPLAINED DEATH (SUD) IN THE YOUNG Jacqueline Neubauer1, Valeria Mereacre1, Giancarlo Russo2, Cordula Haas1

1 Institute of Forensic Medicine- University of Zurich, Forensic Genetics, Zurich, Switzerland 2 Functional Genomics Center Zurich FGCZ, University of Zurich / ETH Zurich, Zurich, Switzerland

Sudden death events in adolescents or young adults often represent the first manifestation of undetected genetic cardiac diseases, which remained without any symptoms during lifetime. Approximately 30 % of these sudden death victims have no definite cardiac etiology after a comprehensive medicolegal investigation and are therefore termed as sudden unexplained death (SUD) cases. Despite of tremendous efforts in the last years to identify underlying genetic diseases in SUD victims, 70–80 % of the SUD cases still remain elusive after genetic testing, emphasizing the importance of additional research to better understand the pathological mechanism leading to a sudden death event. Myocardial transcriptome analysis in human cardiovascular diseases has shown that the RNA expression pattern can clearly be distinguished between patients with different cardiomyopathies and healthy controls. In addition, protein-coding and regulatory RNAs were described to be up- or down-regulated during acute myocardial infarction and heart failures, exhibiting disease-specific expression signatures. The aim of this project was to investigate the RNA expression patterns of heart tissue obtained from postmortem autopsy samples in 34 SUD cases and 17 heart-healthy controls. Whole transcriptome RNA library preparation was performed with the Trio RNA Seq-Kit (NuGEN) and the sequencing was done on the Illumina NovaSeq6000. Hierarchical cluster analysis revealed significant differences in the RNA expression patterns between different subgroups within the SUD cohort and controls.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 199 P119 - NAILS AS A SOURCE OF DNA FOR MOLECULAR GENETIC IDENTIFICATION OF DECOMPOSED HUMAN REMAINS Jezerka Inkret1, Irena Zupanič Pajnič1, Barbara Gornjak Pogorelc1, Jože Balažic1

1 Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

An essential step to achieve identification of decomposed human remains is molecular genetic identification, especially in cases when classical identification methods are not possible. With advanced decomposition, only few tissues can be used as a source of DNA, mostly bones and teeth from which sampling and DNA extraction is difficult and time consuming. In most of highly decomposed cadavers, nails are preserved and can be used instead of bones and teeth. The aim of our study was to evaluate nails as an alternative source of DNA for genetic identification of highly decomposed human remains. Extraction method for quick and optimal gain of sufficient amounts of DNA was optimised and used in 33 routine cases of human identification. The EZ1 Investigator kit (Qiagen) was used for extraction and G2 buffer included in EZ1 kit re-

placed with TNCabuffer. Additionally, DTT was added for lyses of five milligrams of nails. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the NGM kit (TFS).0.2 to 270 ng DNA/ mg of nail was obtained from samples analysed and no inhibition detected, indicating that PCR inhibitors were removed efficiently. Complete STR profiles were obtained from all nails but one where DNA yield was the lowest but still identification was possible since only one locus drop out occurred. Nails are attractive alternative source of biological material for molecular genetic identification of highly decomposed human remains because optimized extraction method is cost effective and less time consuming than the one for obtaining the DNA from bones and teeth and gives us sufficient amounts of DNA for successful STR typing for human identification of highly decomposed cadavers.

200 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P120 - NAPOLEON´S SOLDIERS FROM 1813: DNA ANALYSIS OF LINEAGE MARKERS Jitka Votrubova1, Petr Brestovansky2, Petr Tomasek3,4, Daniel Vanek1,4,5

1 Forensic DNA Service, DNA laboratory, Prague, Czech Republic 2 North Bohemian Museum, Archaeology, Liberec, Czech Republic 3 Institute of Legal Medicine- Bulovka Hospital, Legal Medicine, Prague, Czech Republic 4 Charles University in Prague, 2nd Faculty of Medicine, Prague, Czech Republic 5 Institute of Legal Medicine, Bulovka Hospital, DNA laboratory, Prague, Czech Republic

August 2016 local flooding in Kunratice (suburban of Liberec in North Bohemia) exposed several graves with 15 male individuals. The artefacts found during the excavation suggest that the individuals should belong to soldiers of Napoleon´s army that operated in this region in 1813. DNA analysis of 2 male individuals yielded Y-chromosomal and mtDNA haplotypes. Resulting haplotypes were compared to the contemporary population from the vicinity of the excavation site as well as to the publicly available databases.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 201 P121 - NEW HOPE IN HUMAN IDENTIFICATION CASES: MPS STUDY ON DEGRADED BONE MATERIAL WITH USE OF VEROGEN’S FORENSEQ KIT Maria Szargut1, Marta Diepenbroek2, Grażyna Zielińska1, Joanna Arciszewska1, Sandra Cytacka1, Katarzyna Jałowińska1, Andrzej Ossowski1

1 Pomeranian Medical University in Szczecin, Department of Forensic Genetics, Szczecin, Poland 2 Universität München, Institut für Rechtsmedizin, München, Germany

According to the data collected for the purpose of the GeNN Project (Pol. Genetyczna Identyfikac- ja Zwłok o Nieustalonej Tożsamości, Eng. Genetic Identification of Remains of an Unknown Iden- tity, with NN meaning non notus, Latin for “unknown”), about 800 corpses of people identified as “unknown” are found each year in Poland and a positive identification is obtained for over 85 % of them. Although these statistics may seem like a relatively satisfactory result, by 2015 more than 3 600 human remains, whose identities still haven’t been determined have been buried in Poland. The abovementioned data refers only to corpses found in modern times and does not include victims of armed conflicts. Polish regulations don’t obligate the law enforcement agencies to identify human remains if more than 35 years have passed between the death of a person and the discovery of remains. Meanwhile in Poland, haunted by two World Wars, after which a communist regime time had come, remains of victims of totalitarian systems of the XX century are still being found and the scale of this problem is enormous. With the dawn of MPS era, new tools for human identification arose. The research which we hereby present aims at verification of utility of ForenSeq DNA Signature Prep Kit on MiSeq FGx (Verogen Inc.) for amplification and sequencing of repetitive and flanking regions of autosomal, Y-chromosomal and X-chromosomal STRs, as well as identity-, ancestry- and phenotypically-informative SNP markers for the purpose of personal identification. We examined 75 bone samples, varying in DNA concentration and degradation index coming from different time periods, throughout the XX century (studied within the Polish Genetic Database of Victims of Totalitari- anisms project) and recent times (2015–2017).

202 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P122 - NON-DESTRUCTIVE MEANS OF SAMPLING PAPER DOCUMENTS FOR GENERATION OF STR PROFILES Zoe Wood1, Carl Mayers1

1 DSTL, Porton Down, Salisbury, United Kingdom

Many methods have been published for forensic sampling of touch DNA from a variety of surfaces, with subsequent analysis of collected DNA to generate STR profiles. Comparatively fewer methods exist for sampling touch DNA from porous surfaces, especially paper documents. Ac- cordingly, touch DNA is commonly sampled from documents by simply excising portions of the exhibit. Whilst excision-based sampling generates high-quality DNA profiles, excision also involves partial destruction of evidence. We therefore decided to investigate non-destructive means of sampling paper documents for touch DNA. A scoping study was carried out to identify potential alternative sampling techniques, with a further down-selection exercise on options that showed promise. Sampling using the adhesive portion of a sticky note consistently performed well in these initial stages, generating high-quality profiles without damaging the paper surface. A larger, more robust study was then performed to investigate whether a significant difference in STR profile quality could be found between the existing excision-based method and the new adhesive-based method. Preliminary results indicate no significant difference between the excision- and adhesive-based methods. This indicates that the sticky-note-based method performs as well in the generation of STR profiles from paper as excision, without destruction of the paper exhibit. This new method assists forensic investigators by allowing generation of STR profiles from document exhibits whilst preserving the document as additional evidence. © Crown copyright (2019), Dstl. This material is licensed under the terms of the Open Govern- ment Licence (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 203 P123 - NON-INVASIVE PRENATAL PATERNITY TESTING BY MATERNAL PLASMA DNA SEQUENCING IN TWIN PREGNANCIES Yifan Xie1, Fang Chen1, Yanyan Zhang1, Yicong Wang1, Guifang Yang1, Xueling Ou2

1 MGI-BGI-Shenzhen, Application research and development center, Shenzhen, China 2 Sun Yat-sen University, Faculty of Forensic Medicine, Zhongshan School of Medicine, Guangzhou, China

Routine prenatal paternity testing was performed by chorionic villus sampling or amniocentesis combined with short tandem repeats(STRs) based on capillary electrophoresis (CE). The invasive procedure may cause a potential miscarriage risk for pregnant women, and the late sampling time delayed the decision for pregnancy outcome. Non-invasive approaches based on single-nucleotide polymorphisms(SNPs) and next-generation sequencing (NGS) have been investigated in single pregnancies for years. However, the feasibility in twin pregnancies remains uncertain, mainly due to more complex DNA profile of maternal plasma in these cases. Here we described a non-invasive method based on maternal plasma target SNP sequencing to determine the paternity of twin pregnancies. Fifteen families (twin pregnancies at 9 to 18 weeks of gestation) were recruited and analysed with custom-designed probes covering 5011 SNPs based on Illuminaplatform.A mathematical model for data interpretation was established, including zygosity determination and paternity index calculations. Each plasma sample was independently tested against alleged father and 90 unrelated males. Paternity was confirmed by genetic testing of the foetal tissues in a blinded manner. After quality control, the combined paternity index (CPI) for alleged father and 90 unrelated males were calculated. In the 15 twin pregnancies, including 8 monozygotic and 7 dizygotic twins, the correct biological father was successfully identified, and the paternity of all 90 unrelated males were excluded. Our study first demonstrated that non-invasive prenatal paternity testing could be performed for twin pregnancies using target sequencing of maternal plasma without advanced knowledge of zygosity.

204 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P124 - NOVEL INDEX OF BODY FLUID-RNA INTEGRITY BASED ON SMALL RNA ELECTROPHEROGRAM Shuntaro Fujimoto1, Sho Manabe1, Eriko Hirai1, Chie Morimoto1, Keiji Tamaki1

1 Kyoto University Graduate School of Medicine, Forensic medicine, Kyoto, Japan

Measuring RNA integrity is indispensable for performing experiments with RNA because RNA molecules are labile. RNA integrity number (RIN) is widely used in various fields of RNA experiments. RIN is calculated based on high molecular peaks and low molecular peaks in a total RNA electropherogram. RIN for pristine body fluids RNA is sometimes very low despite good quality RNA. We previously reported that results from small RNA electropherograms correlated well with RNA quality from body fluids. In this study, we aim to develop a new index of RNA integrity for body fluids. We used six pristine venous blood samples for model building, and some pristine body fluids and postmortem blood for a test. Total RNA was extracted from the samples using the mirVanaTM microRNA Isolation Kit. These RNAs were fragmented by ultraviolet (UV) irradiation for 4–40 minutes. Small RNA electrophoresis was performed with the Agilent small RNA kit by the Agilent 2100 Bioanalyzer. Data were collected using Agilent 2100 Expert software ver. B.2.08.SI648. RNA integrity index was developed based on partial least squares regression (PLS) and time-series analysis of UV exposure to RNA by use of RNA electropherograms. We found that PLS component 1 increased with increased exposure UV irradiation. The RNA quality of pristine body fluids was high and correlated well with PLS component 1 although RIN for these fluids was low. However, postmortem blood showed various PLS component 1 regardless of postmortem intervals of the samples. In conclusion, the index was effective for measuring RNA integrity in pristine body fluids, but not for RNA in various states of degradation. In future, we would demonstrate the utility of the index in various degraded state of samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 205 P125 - NOVEL TRI-ALLELIC PATTERN OF THE FGA MARKER Viktor Poór1, Vivien Fejes1, Dominika Szűcs1, Zsolt Kozma1, Zsolt Pádár1

1 University of Pécs- Medical School, Department of Forensic Medicine, Pécs, Hungary

Here we report an interesting case of tri-allelic FGA mutation. During database creating a sample produced a tri-allelic pattern on the FGA locus resulting alleles of 21.2, 22 and 23.2. Moreover, the peak heights suggest the presence of four alleles. This result was reproduced with Investi- gator ESSplex SE Plus (Qiagen) and AmpFlSTR NGM SElect (Thermo Fisher Scientific) kits from both blood and buccal swab samples. We did not observe any other discrepancies of the tested markers. We were able to recruit the parents. The observed alleles of the mother were 23 and 23.2 while the father was homozygous 22. All the 16 markers were in accordance with the paternity. Occurrences of tri-allelic patterns of several STR markers (e.g. TPOX, vWA, Penta D, D21S11), are reported, but none of the FGA marker.

206 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P126 - OMPTIMISATION AND COMPARISON OF MUTIPLEX mRNA-BASED BODY FLUID IDENTIFICATION BY CAPILLARY ELECTROPHORESIS (CE) AND MASSIVE PARALLEL SEQUENCING (MPS) Malte Bamberg1, Galina Kulstein2, Angelika Fürst3, Peter Wiegand1, Thorsten Hadrys3

1 Institute of Legal Medicine, Forensic Genetics, Ulm, Germany 2 Federal Criminal Police Office, KT31- DNA-Analytics, Wiesbaden, Germany 3 Bavarian State Criminal Police Office, DNA Department, Munich, Germany

The identification of the origin of biological material of human body fluids and tissues of stains found at crime scenes are crucial in the context of the circumstance of crime. Therefore, especially mRNA-based body fluid and tissue identification (BFI) has been vigorously examined in the last decades. Besides Reverse Transcriptase (RT) capillary electrophoretic (RT-CE)- and quantitative PCR (RT-qPCR)-based approaches, also massively parallel sequencing (MPS)-based approaches, such as a targeted multiplex analysis of 33 RNA markers, have recently been introduced for mRNA profiling. In this study, we i) present the optimisation of RT-CE- and MPS-based approaches for BFI. Optimisation was conducted by adjustment of primer concentrations as well as by replacement/removal of insen- sitive markers. ii) discuss the results of the comparison of the performances between the RT-CE- and MPS- based approaches for BFI in regard to robustness, sensitivity and specificity. For the comparison, we included forensic relevant body fluids/tissue such as blood, saliva, vaginal and menstrual secretions, semen and skin. We also considered genuine body fluid mixtures as well as samples from routine casework. All in all – though both approaches consist of different sets and amounts of RNA markers – the results concerning body fluid origin and the amount of correctly observed body fluids are reliable concordant for both methods.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 207 P127 - OPTIMIZATION AND APPLICATION OF A LARGE SNP PANEL UTILIZING UNIQUE MOLECULE INDICES FOR MISSING PERSONS IDENTIFICATIONS Michelle Peck1, Felix Bittner1, Šejla Idrizbegović1, Ana Bilić1, René Huel1, Christopher Phillips2, Andreas Tillmar3,4, Thomas Parsons1

1 International Commission on Missing Persons, Science and Technology, The Hague, Netherlands 2 University of Santiago de Compostela, Institute of Forensic Sciences, Santiago de Compostela, Spain 3 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden 4 Linköping University, Department of Clinical and Experimental Medicine, Linköping, Sweden

A large SNP panel (“MPSplex”) consisting of over 1200 SNP targets was designed to address two of the biggest challenges with missing persons cases – highly degraded DNA and the need for kinship matching based on single and/or distant relatives. In addition to highly heterozygous, tri-allelic SNPs, the MPSplex panel includes 55 ancestry-informative SNPs and 45 microhaplotypes. The assay is based on QIAGEN’s QIAseq chemistry that uses Unique Molecular Indices (UMIs) that tag the original DNA molecules represented in the library, imparting substantial advantages in data interpretation and establishing thresholds. Additionally, target enrichment utilizes single target-specific primers, decreasing the difficulties posed by dual primer PCR. Sensi- tivity studies with control DNA and challenging bone extracts indicate that powerful amounts of genetic data can be obtained from low quantity and quality DNA. Even 31 pg of degraded DNA produced data with as much kinship power as multiple STR profiles. Calling thresholds for homozygote and heterozygote loci have been explored in relation to UMI coverage and sequence quality scores, and first approaches to deal with drop-in and drop-out will be presented. Overall, the power and sensitivity of the assay significantly expands the range of missing persons cases that can be solved by forensic genetics. In some samples with DNA fragment sizes ~150bp or less, over 1000 SNPs were typed, when only a few STR alleles were recovered. In initial case applications on degraded bone samples, ICMP has reported highly significant DNA matches with first cousins as references. Further developments to improve the throughput of the workflow will be critical to taking full advantage of the power of this method in the most difficult missing persons cases.

208 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P128 - OPTIMIZING THE PROCESSING OF DATABASING SAMPLES USING THE HAMILTON EASYPUNCH® STARLET Dan Watsula1, Allie Flores1, Heather Sarik2, Kevin W.P. Miller3, Robert Bever1, Kristen Naughton2, Manzar Ahmed1

1 Bode Technology, Products, Lorton, USA 2 Bode Technology, Validations, Lorton, USA 3 Hamilton Robotics, Senior Market Segment Leader, Reno, USA

The passing of legislation allowing for the collection of DNA samples from arrestees as well as all population databases has led to some laboratories facing a significant increase in the number of samples submitted. This has the potential to create a backlog situation for the DNA Database analysts. Direct amplification of reference samples has increased efficiency compared to the traditional method but still requires staff to either manually remove 1.2 mm punches or manually feed a semi-automated instrument. Subsequently, the analyst or technician must perform the amplification setup as a separate process and procedure. This study evaluated the Hamilton easyPunch STARlet to improve the efficiency and throughput of a databasing section. The easyPunch STARlet is designed as an “all-in-one” system that will provide both sample punching and liquid handling. Three direct amplification chemistries were evaluated as part of this study; ThermoFisher’s Globalfiler™ Express, Promega’s PowerPlex® Fusion 6C, and QIAGEN® Investigator 24plex GO!. Ninety (n=90) Bode Buccal 2 collected samples were processed with each amplification kit. The resulting DNA profiles were analyzed using appropriate laboratory analytical and stochastic thresholds. The data metrics evaluated included first amplification success rate, average locus peak height, and average intra-color balance. The optimized protocol provided a method to obtain a 96-well plate containing both lysed sample punches and amplification master mix in under 2 hours with minimal human interaction. 99 % of the samples produced a complete DNA profile upon first amplification. The results from this study show that a fully automated platform can increase a laboratory’s efficiency without decreasing profile quality or success rates.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 209 P129 - PARALLEL SEQUENCING OF 48 Y-CHROMOSOME STR AND SNP MARKERS Ruiyang Tao1,2, Wei Song3, Chong Chen2,4, Jiashuo Zhang2,5, Jingyi Zhang2,5, Suhua Zhang2, Chengtao Li1,2

1 Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Forensic Genetics, Chengdu, China 2 Academy of Forensic Science, Ministry of Justice- China, Forensic Genetics, Shanghai, China 3 Minhang Criminal Police, Shanghai Public Security Bureau, Forensic Genetics, Shanghai, China 4 College of Medicine and Forensics, Xi’an Jiaotong University Health Science Center, Forensic Genetics, Xi’an, China 5 Medical School of Soochow University, Forensic Science, Suzhou, China

Over the last decade, massively parallel sequencing (MPS) has been developing rapidly, bringing many advantages to forensic genomics. It could simultaneously target multiple genetic markers including short tandem repeats (STRs), insertion and deletion polymorphisms (InDels), and single nucleotide polymorphisms (SNPs). In this study, we developed an assay which can simultaneously sequence 22 Y-STRs and 26 Y-SNPs on the MiSeq FGxTM Forensic Genomics System (Illumina, Inc., San Diego, USA). The primers of the amplicons were designed using the online De- signStudio (Illumina). Subsequently, DNA libraries were established with the TruSeq method. For the pilot study, we sequenced 25 unrelated Chinese Han males. A total of 105 sequence-based alleles were observed at the 22 Y-STR loci, with the average depth of coverage (DoC) ranging from 314.9 x to 637.7 x. For the concordant study, the sequencing results of these samples were fully consistent with genotypes called via capillary electrophoresis (CE). For the SNPs, a preferable performance was observed with the average DoC of 1170.2 x. However, inter-locus imbalance were observed at some of the markers, which needs substantial improvement of the primer pool. In general, this customized Y-chromosome STR and SNP panel on the MiSeqFGxTM Forensic Genomics System provides an effective solution for these markers on Y chromosome, which may allow more forensically relevant information to be obtained from limited forensic materials, such as those from sexual assault and rape cases. Besides, it could also facilitate the forensic practice including paternity testing, genetic determination of pedigree and inter-population migration integration.

210 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P130 - PETROUS BONE: AN OPTIMAL SUBSTRATE IN LEGAL MEDICINE? Angéla Gonzalez1, Catherine Cannet1, Vincent Zvènigorosky2, Annie Geraut3, Guillaume Koch4, Tania Delabarde2, Bertrand Ludes2, Jean-Sébastien Raul1, Christine Keyser1

1 Institut de Médecine Légale, Université de Strasbourg, Strasbourg, France 2 Institut Médico-Légal de Paris, CNRS FRE2029 Université Paris Descartes, Paris, France 3 Institut de Médecine Légale, Les Hôpitaux Universitaires de Strasbourg, Strasbourg, France 4 Institut d’Anatomie Normale, Les Hôpitaux Universitaires de Strasbourg, Strasbourg, France

Over the last five years studies in paleogenomics have shown that the petrous bone, the densest part of the temporal bone, is capable of providing endogenous DNA of superior quantity and quality compared to other bony substrates. If this dense part, corresponding to the bone lab- yrinth, also known as the otic capsule, is considered the substrate of choice in paleogenomics, it could be considered equally as important in forensic science to identify individuals based on skeletal remains. There are two main objectives in this study : (I) to better understand why the petrous bone is the substrate of choice for ancient DNA analysis and (II) to establish if the substrate delivers well-preserved DNA each time. To obtain our answers we performed histological analyses (I) to compare the bone structure of a petrous bone to that of a tooth, and (II) to compare a petrous bone taken from an archaeological or forensics sample to that of a fresh petrous bone. At the same time, using 30 samples of archaeological origin and 11 forensic samples, we analysed the STR autosomal profiles obtained from the petrous bone and tooth in order to compare the quality of the DNA taken from these two substrates. Histological analyses revealed microstructural characteristics (a peculiar tissue organization) unique to the petrous bone which could explain how the DNA molecules are so well-preserved. Statistical calculations made from the amplified STR loci show that the petrous bone contains a higher quantity of DNA molecules than the tooth, improving our chances of obtaining an STR profile in genetic identification. Even if the otic capsule is damaged from the diagenesis process, it’s utilization in forensics could prove valuable, especially in cases involving infants or toothless individuals.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 211 P131 - PILOT IDENTITY PROJECT. MISSING BUT NOT FORGOTTEN – IDENTIFICATION OF THE POLISH AIR FORCE IN GREAT BRITAIN PILOT MIA IN 1943 Andrzej Ossowski1, Joanna Arciszewska1, Grażyna Zielińska1, Iwona Teul1, Jan Ambroziak2, Sandra Cytacka1, Maria Szargut1, Magdalena Buś3

1 Pomeranian Medical University in Szczecin, Department of Forensic Genetics, Szczecin, Poland 2 The Ministry of Culture and National Heritage, The Ministry of Culture and National Heritage, Warszawa, Poland 3 University of North Texas, Center for Human Identification, Fort Worth, USA

The case presented here is an example that genetic methods backed up by extensive archival research are effective in identification of unnamed remains from World War II. Human identification of remains from mass disasters including armed conflicts is demanding. Usually, a number of victims are involved and limited information about their fate is available. Recently, more attention has been paid to identification of soldiers killed in action during numerous armed conflicts. In an identification study, the key role is attributed to DNA analysis. DNA analysis with the use of STR markers is routinely used for human identification both in ordinary criminal cases and identification of mass-disasters victims. In the case of victims of totalitarian systems and war conflicts, especially World War II, such studies are conducted incidentally. DNA preserved in aged remains is severely degraded and present in a low copy number; therefore, achieving genetic information may not be possible or only partial STR profiles will be obtained. This can lead to failure of the identification process. In the study, we present an example of individual identification of a Polish pilot serving in the RAF and taking part in the Battle of Britain, MIA in 1943 after his fighter plane fell into the English Channel on the return way from Northern France land target harassment mission. The case was complex and involved many challenges in the identification of the searched person. The final identification was possible by exploration of official documents and combining the expertise of several specialists from the fields of history, anthropology, and genetics.

212 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P132 - POPULATION GENETIC DATA FOR 27 AUTOSOMAL STR MARKERS IN THE KUWAITI POPULATION USING MASSIVELY PARALLEL SEQUENCING Mahdi Haidar1,2, Mark A. Jobling3, Yahya M. Khubrani3, Jon Wetton3, Penelope R. Haddrill1

1 University of Strathclyde, Centre for Forensic Science, Department of Pure and Applied Chemistry, Glasgow, United Kingdom 2 Kuwait Identification DNA Laboratory, General Department of Criminal Evidence, Ministry of Interior, Kuwait City, Kuwait 3 University of Leicester, Department of Genetics & Genome Biology, Leicester, United Kingdom

Recent years have seen the introduction of massively parallel sequencing (MPS) in forensic genetics, a DNA sequencing technique that out-performs the current dominant PCR-CE typing system of forensic STR and SNPs markers. In particular, MPS can detect the actual nucleotide sequences of STR alleles, rather than just their length. This substantially increases the information obtained from samples, and thus the discrimination power of forensic testing, as well as improving mixture interpretation and assisting in paternity testing. It is anticipated that MPS will become increasingly used in forensic laboratories. However, for the sequencing system to be implemented in laboratories, population studies must be carried out to determine allele frequencies, which facilitate the calculation of statistics to estimate the strength of DNA profile evidence and to assess the performance of each marker assayed using MPS. To date, no previous study has investigated MPS of autosomal STRs in the Kuwaiti population. This work aims to evaluate the forensic utility of 27 autosomal STRs markers contained in the Verogen ForenSeq™ DNA Signature Prep Kit using the MiSeq FGx instrument in a sample of 370 unrelated Kuwaiti individuals. The results showed an increased number of genetic variants detected for these markers, based on their sequence rather than their length. This suggests these markers will be highly informative, and that this system could be successfully used for individual identification and paternity testing in the Kuwaiti population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 213 P133 - POPULATION STRUCTURE AND THE CONSERVATION STATUS OF THE ROUGH-TOOTHED DOLPHINS BASED ON THE ANALYSIS OF THE MITOCHONDRIAL CONTROL REGION Anna Donato1, Salvatore Siciliano2, Marcelo Weksler3, Dayse Silva4, Elizeu Fagundes Carvalho4, Silvia Martins5, Cesar Amaral4

1 Universidade Federal do Rio de Janeiro - Museu Nacional, Departamento de Vertebrados, Rio de Janeiro, Brazil 2 Fundação Oswaldo Cruz - Instituto Oswaldo Cruz, Laboratório de Enterobactérias, Rio de Janeiro, Brazil 3 Universidade do Estado do Rio de janeiro - Museu Nacional, Departamento de Vertebrados, Rio de Janeiro, Brazil 4 Universidade do Estado do Rio de Janeiro, Departamento de Ecologia - Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 5 Universidade do Estado do Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil

The rough-toothed dolphin (Steno bredanensis) is found in deep waters in tropical, subtropical and warm temperate regions of the Atlantic, Pacific and Indian Oceans. In Brazil, however, they can be observed in coastal waters from Pará to Rio Grande do Sul, a habit that makes them susceptible to several anthropogenic impacts. This species is one of the small cetaceans with the most recorded cases of accidental catches in fishing nets (bycatch). In the south-eastern and southern regions of Brazil, between 1987 and 1997, several accidentally captured individuals were observed. These animals had markings of fishing nets on their body, severe mutilations, wounds caused by blows of sharp / piercing instruments and pieces of nets and ropes attached to the body. Although Steno bredanensis is classified as ‘least concern’ by the IUCN RedList, little is known about its population size and population structure. Because they show evidence of site fidelity, it is important to delimit populations to assess the actual conservation status of the species and the impact of fishing activities on it. The present study evaluates the genetic structure of rough-toothed dolphins based on individuals from Rio de Janeiro, Brazil using the mitochondrial D-Loop region and discuss the present conservation status of the species related to the fishing activities conducted along the Brazilian coast. Keywords: Steno bredanensis; mitochondrial D-Loop; dolphins.

214 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P134 - POST MORTEM DNA ANALYSIS OF DECAYED HUMAN REMAINS – PART 1/4: A RETROSPECTIVE OF UNKNOWN HUMAN BODIES AT THE INSTITUTE OF LEGAL MEDICINE BASEL (2014–2019) Alina, Senst1, Iris Schulz1

1 Institute of Legal Medicine Basel, PhD Student, Basel, Switzerland

Deceased persons are usually identified by fingerprints or dental status. In cases where no such data is available, the ultimate tool for identifying the unknown body is DNA analysis. Despite advances in modern DNA analysis, it is still a challenge to identify bodies using short tandem repeats (STRs) that have undergone major changes in decay. In such cases, the question can arise as to which material results in complete DNA profiles at the first attempt. The aim of the project is to develop recommendations for the efficient DNA identification of altered human remains. We investigate which material with different stages of decay is best suited for corpse identification using the current DNA marker sets and different laboratory platforms. In this poster we present the first part of the studies:A retrospective on the identification of decayed human bodies at the Institute of Forensic Medicine in Basel (2014–2019). The result will provide the number and type of corpses as well as the repetition rate of the analysis and the success rates of the identification. Beside the outcome of (1) the retrospective study, we will present the concept of the larger study: (2) the collection of potential material (i.e. soft and hard tissue) from different types of corpses over a period of three years and associated STR analysis; (3) MPS-based analysis to include phenotypic and biogeographic approximations; (4) the evaluation of the robustness of additional DNA methylation markers by testing different storage conditions and tolerance to potential inhibitors on MPS analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 215 P135 - PRECISION ID GLOBALFILER MIXTURE STUDY Torben Tvedebrink1, Søren Byg Vilsen1, Helle Smidt Mogensen2, Poul Svante Eriksen1, Niels Morling2

1 Aalborg University, Department of Mathematical Sciences, Aalborg, Denmark 2 University of Copenhagen, Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, Copenhagen, Denmark

The Precision ID Globalfiler NGS STR panel (Applied Biosystems, ThermoFisher Scientific) analyses 31 autosomal STRs, four Y- and one X-chromosomal markers by Massively Parallel Sequenc- ing (MPS) using the Ion Torrent semiconductor technology. We analysed a total number of 72 female:male DNA mixtures with ratios of 1:1, 3:1 and 10:1 with a total amount of input DNA of 500, 250 and 125 pg on an Ion Torrent S5. The marker specific STR regions were identified using the flanking regions. A statistical Pois- son-gamma coverage model was used to estimate the mixture proportions and marker imbalances of each sample, given the profiles major and minor contributors. Similar to prior experience, the MPS results showed a large between-marker variation. Eight alleles and four markers dropped out across the 72 samples (allele coverage of zero). One allele dropped out in a 250 pg sample and the remaining in the 125 pg samples. All drop-outs was due to the minor contributor in 10:1 mixtures. As expected, the variance of the mixture proportions was largest for 1:1 mixtures, with increased variation as the amount of input DNA decreased (less pronounced for 3:1 and 10:1 mixture samples).

216 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P136 - PRELIMINARY EVALUATION OF FORENSIC CASEWORK SAMPLES USING THE PRECISION ID ANCESTRY PANEL – MANUAL AND AUTOMATED AMPLISEQ WORKFLOW Waad Aldosari1, William Goodwin2, Ibrahim Al Binali3, Satya Sudheer Pydi4

1 Ministry of Interior- Forensic Laboratory Department.Qatar, School of Forensic and Applied Sciences- University of Central Lancashire. Preston- UK, Doha, Qatar 2 University of Central Lancashire- Preston- UK, School of Forensic & Applied Sciences, Preston, United Kingdom 3 Ministry of Interior, Forensic Laboratory Department, Doha, Qatar 4 Sedeer Medical, Application Team Lead, Doha, Qatar

The Precision ID Ancestry panel from ThermoFisher Scientific comprises 165 AI-SNPs. The panel was used to analyze 143 biological evidence samples, collected between 2005 and 2018, in the State of Qatar. The samples varied from routine stains with high amounts of DNA to challenging samples that had yielded partial profiles when analyzed using STR kits, such as AmpFl- STR Identifiler and GlobalFiler. Two Ampliseq workflows were used: manual and automated library preparation, using the Ion Chef, followed by sequencing on an Ion PGM instrument. The samples were amplified based on the DNA concentration with the standard 21 cycles used for samples where >1.5 ng DNA could be added and 25 or 26 cycles for samples below 1.5 ng template. The manual workflow produced 47 full profiles, 53 partial profiles, and 11 fails; the automated workflow produced 29 full profiles, 18 partial profiles, and 1 fail. Within these samples 16 were analyzed using both workflows; no significant differences were seen between the two workflows. However, automated workflow saved considerable hands-on lab time. The ancestry prediction was undertaken using HID SNP Genotyper v5.2.2 as Admixture Predic- tion and Population Likelihood. As expected, most samples were predicted to be of Middle East- ern or South Asian origin, but others predicted to be African, European, and East Asian.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 217 P137 - PRESERVATION OF DNA INTEGRITY IN BIOLOGICAL MATERIAL Adria Burrows1, Maria Eugenia D’Amato1

1 University of the Western Cape, Biotechnology, Cape Town, South Africa

The collection and preservation of biological material is critical to many biomedical and ecology fields. Samples need to be preserved so that DNA or RNA analysis can be performed once samples reach the research/ medical facilities. The FDL experienced deterioration of samples and degradation when collecting buccal swabs in remote areas with limited or devoid of cold storage facilities. To overcome this problem in 2015 we designed a buffer that allows storage of human saliva at room temperature for two years, protecting the DNA from degradation [1]. The project has been expanded to test the preservation ability of the buffer with other types of biological material: plant material, fish fins and human blood samples. In this work, we analyse saliva preserved in the buffer at room temperature for four years. The extracted DNA was quantified with Investigator® Quantiplex (Qiagen) and genotyped using GlobalFiler™ PCR Amplification Kit. The newly tested plant and animal biological materials were kept at room temperature for six months. The genomic DNA of the different biological material samples were extracted using specific extraction methods for the sample type. The quantity and quality of the DNA was evaluated with Nanodrop ND-2000 UV spectrophotometer and agarose gel electrophoresis. The quality of the extracted DNA was further evaluated by sample specific PCR. References [1] Burrows, A. M., Ristow, P. G., & D’Amato, M. E. (2017). Preservation of DNA from saliva samples in suboptimal conditions. Forensic Science International: Genetics Supplement Series, 6(Septem- ber), e80–e81. https://doi.org/10.1016/j.fsigss.2017.09.050.

218 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P138 - PREVALENCE OF DNA IN VEHICLES: LINKING AN ITEM AWAY FROM A VEHICLE TO OCCUPANCY OF THE VEHICLE Toni Boyko1, Bas Kokshoorn2, Roland van Oorschot1

1 Victoria Police Forensic Services Centre, Office of the Chief Forensic Scientist, Macleod, Australia 2 Netherlands Forensic Institute, Division of Biological Traces, The Hague, Netherlands

It is important to improve our understanding of several aspects relating to DNA transfer, persistence, prevalence and recovery (DNA-TPPR) to assist investigations of crime. One such aspect is the extent to which a person picks up DNA from one location and transfers it to another. In this study a known temporary driver drove a car, regularly driven by a known non-associate, for ~30 min along a prescribed route. A previous study of these cars and temporary drivers, demonstrated that DNA originating from the original driver of a car can be detected on the clothing of the temporary driver after vacating the car. As part of the current study the temporary driver of each car was also asked to place the used keys in a clean petri dish on a nearby table, immediately after parking and vacating the car. The temporary driver was also asked to subsequently firmly place their left and right hand onto separate DNA-free glass plates. Samples were taken from the glass plates and keys within 5 min of creating the handprints. This was repeated for 15 different car/temporary driver combinations. Sufficient DNA to generate reportable profiles was recovered from the majority of handprints and all keys. Here we report on the detection of the temporary driver, regular driver, known associates of the temporary driver, known associates of the regular driver, and unknown contributors, within the reportable profiles generated from the handprints and keys. These results demonstrate that DNA of a regular driver of a car can be picked-up by a temporary driver and transferred to a location beyond the car.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 219 P139 - PREVALENCE OF DNA IN VEHICLES: PERSISTENCE OF DNA FROM A TEMPORARY DRIVER AFTER REUSE BY ITS REGULAR DRIVER ON ITEMS LESS COMMONLY TARGETED Toni Boyko1, Bas Kokshoorn2, Roland van Oorschot1

1 Victoria Police Forensic Services Centre, Office of the Chief Forensic Scientist, Macleod, Australia 2 Netherlands Forensic Institute, Division of Biological Traces, The Hague, Netherlands

It is relevant to investigators of crimes to have an understanding of the prevalence and persistence of DNA from a temporary user of an item, or items within a space. This is so when there has been no further use of the items, as well as after they have been reused by the usual user. A previous study has investigated this in relation to the use of cars, where 20 core sites (including relating to: seats, seatbelts, steering wheel, interior door arm, gear lever, handbrake, rear-view mirror, exterior glove box panel) were sampled from 10 different cars regularly driven by a known single driver at different time intervals (immediate, 1 day, 1 week) after having been driven (for 30 min) by a different known temporary driver on each occasion. Samples were collected from a further 13 non-core sites within a subset of cars of the previous study. These sites were chosen as they would likely, or could possibly, be contacted by a temporary user of a car. They include sites relating to: exterior and interior door handles, window switch, exterior mirror adjuster, driver backrest adjuster, indicator/ wiper/ light levers, dashboard temperature and sound control buttons, mid-caddy armrest and opening lever. We report here on the quantity of DNA retrieved, the number of contributors to the profiles generated, the types of profiles, the relative presence of the regular driver, temporary driver, their known close associates (such as their co-resident partner) and other unknowns. We also compare the results from these sites with those of the core set. The results of this study will inform us on the transfer, persistence, prevalence and recovery of DNA from a wider array of sites within the front compartment of vehicles. This will assist sample targeting and assessment of considered events.

220 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P140 - QUANTITATIVE EVALUATION OF DNA ISOLATED FROM BACKSPATTER RECOVERED FROM FIREARMS AND WOUND PROFILE CHARACTERISTICS AS EFFECTS OF SHOOTNG DISTANCE Jan Euteneuer1, Annica Gosch1, Philipp Cachée2, Cornelius Courts1

1 University Medical Center Schleswig-Holstein, Institute of Forensic Medicine, Kiel, Germany 2 Sachverständigenbüro Cachée, Forensic Ballistics, Berlin, Germany

When a biological target is hit by a bullet, tissue fragments and blood are propelled out of the entrance wound and back into the direction of the gun by wound ballistic effects, a phenomenon termed ‘backspatter’. These biological traces can hold clues for the identification of victims from gun-related crimes as well as for the determination of wounding area and severity via identification of organ tissue and/or body fluids, thereby contributing to the evidence-based reconstruction of gun-related crimes. It has been demonstrated that backspatter traces can even consolidate and persist on most inner parts and surfaces of a fired gun. In this study, to assess the influence of shooting distance on the distribution and quantity of backspatter recoverable from inside the gun, we employed a previously developed anatomical- ly realistic skull model, with a gelatin core doped with blood and contrast agent, in a series of shooting experiments involving a pistol and a revolver representing the most frequently used types of firearms in gun-related homicides with the occipital bone as the typical target region. Shootings were conducted in triplicates per shooting distance for each weapon, starting with contact shots and increasing stepwise. After each shot, backspatter traces were collected from several inside surfaces of the gun and DNA was quantified. Wound profile assessment was performed, first by radiological imaging of the skulls via CT and then by optical analysis, involving serially cutting the gelatin core into thin slices and applying the polygon measurement method. Both DNA yield and wound profiles were investigated in relation to the shooting distance, correlated to one another and evaluated for potential patterns and limits.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 221 P141 - RECOVERY OF HOMININ DNA FROM PLEISTOCENE SEDIMENTS AT DENISOVA CAVE Elena Zavala1, Cesare de Filippo1, Frédéric Romagné1, Viviane Slon1, Anatoly Derevianko2, Michael Shunkov2, Maxim Kozlikin2, Zenobia Jacobs3, Bo Li3, Kieran O’Gorman3, Richard Roberts3, Svante Pääbo1, Matthias Meyer1

1 Max Planck Institute for Evolutionary Anthropology, Genetics, Leipzig, Germany 2 Russian Acadamy of Science, Archeology and Ethnography, Novosibirsk, Russian Federation 3 University of Wollongong, School of Earth and Environmental Sciences, Wollongong, Australia

The analysis of DNA sequences has become an important tool for both reconstructing the evolutionary history of humans and other species and forensic casework. Environmental DNA from sediments has been utilized in both the fields of ancient DNA and forensic science to either increase understanding of an archaeological site or link evidence to crime scenes. In 2017, the ability to recover hominin mitochondrial (mt) DNA sequences from Pleistocene sediments was demonstrated (Slon et al, 2017). In concert with a fully automated pipeline from DNA extraction purification (Rohland et al, 2018), library preparation, qPCR and mtDNA capture this has increased the number of samples and sites that may be screened for ancient hominin DNA preservation. This study presents the methods and analysis used for the first high-resolution profiling of sediment DNA at an archaeological site, Denisova Cave in the Altai Mountains in Russia. The potential application of these methods to forensic casework will also be discussed.

222 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P142 - ROOM TEMPERATURE STORAGE OF TISSUE SAMPLES (BOVINE) IN READILY AVAILABLE MEDIA DURING MASS FATALITY INCIDENTS Xavier Chan1, Guo Wei Kua1, Michelle Lai1, Holden Lim1, Ming Xue Wee1, Christopher Kiu Choong Syn1

1 Health Sciences Authority, Biology Division, DNA Profiling Laboratory, Singapore, Singapore

In a mass fatality incident (MFI), where severe body fragmentation may occur, DNA profiling is often the only primary identifier available for re-association and identification of the remains. Singapore has a typical tropical climate with abundant rainfall, high and uniform temperature (mean daily > 27 °C; mean daily max > 30 °C), and high humidity (mean RH of 83 %) all year round. Under such environmental conditions, without proper sample preservation, the success rate of obtaining a complete DNA profile from the remains can be expected to be low. Good preservation of tissue samples can be achieved through storage in -20 °C to -80 °C freezers. How- ever, depending on the scale of the incident, there may be insufficient freezers available on site or at a short notice. Such situations would be exacerbated during national emergencies where electrical shortages may even be encountered. In this study, we investigated the possibility of preserving tissue samples in readily available media, such as kitchen salt and drinking liquor, at room temperature over a period of 24 months. As human samples were inaccessible, bovine samples were used as proxies. Phase 1 data, up to 3 months’ storage, suggests that kitchen salt is a good alternative for tissue preservation as full bovine DNA profiles (up to 250 bp) are readily recovered.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 223 P143 - SCREENING FOR CPG SITES ADJACENT TO COMMON SNPS FOR BLOOD-SPECIFIC GENOTYPING FROM MIXED BODY FLUID SAMPLES Ken Watanabe1, Kei Taniguchi1, Tomoko Akutsu1

1 National Research Institute of Police Science, First Department of Forensic Science, Kashiwa, Japan

Genotyping from mixed body fluid samples is a major problem in forensic investigations. Recent- ly, we reported a novel approach for semen-specific genotyping that can detect semen-specific methylated or unmethylated CpGs and neighboring single nucleotide polymorphisms (SNPs) on the same DNA molecule by combining methylation-specific PCR (MSP) and pyrosequencing. This approach was applied successfully to mixed DNA and may be useful for other body fluid types, such as blood, that are difficult to physically separate. In this study, we applied this approach to blood-specific genotyping by screening for blood-specific CpGs adjacent to common SNPs. Genome-wide methylation was assessed by HumanMethylation EPIC (Illumina) using two sets of pooled samples of blood, semen, saliva, and vaginal swabs. More than 100 CpGs were identified as candidate blood markers using the following thresholds: average methylation ratio of blood (>0.3) and maximum methylation ratio in non-blood samples (<0.1). Among the identified CpGs, five CpGs adjacent to common SNPs (minor allele frequency >0.2) were evaluated for blood-specificity along with neighboring CpGs in each region. Bisulfite sequencing using the MiSeq platform (Illumina) for DNA from blood, semen, saliva, and vaginal fluid (n=4) revealed blood-specific methylation of several, but not all, CpGs in each region. Not all methylated adjacent CpGs were blood-specific, indicating MSP was not appropriate for blood-specific genotyping in these regions because of the difficulty in designing primers. Instead, the targeted bisulfite sequencing we used in this study may be a better choice because this method can perform multiplex methylation analysis at single-CpG resolution and simultaneously analyze adjacent SNPs on the same molecule.

224 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P144 - SEMI-AUTOMATED AUDITING AND ANNOTATION OF REFERENCE DATABASES OF DNA BARCODES FOR SPECIES IDENTIFICATION Eduardo Conde-Sousa1,2, Nádia Pinto3,4,5, António Amorim3,5,6

1 Instituto de Investigação e Inovação em Saúde i3S- Universidade do Porto- Portugal, Bioimaging, Porto, Portugal 2 Instituto de Engenharia Biomedica INEB- Universidade do Porto, Bioimaging, Porto, Portugal 3 Institute of Pathology and Molecular Immunology from University of Porto IPATIMUP, Population Genetics and Evolution, Porto, Portugal 4 Center of Mathematics of the University of Porto, Center of Mathematics of the University of Porto- Portugal, Porto, Portugal 5 Instituto de Investigação e Inovação em Saúde i3S- Universidade do Porto- Portugal, Population Genetics and Evolution, Porto, Portugal 6 Faculty of Sciences of the University of Porto, Biology, Porto, Portugal

Reference databases, such as BOLD or GenBank, are indispensable tools for genetic based taxonomic identification of specimens. Millions of DNA barcodes with associated metadata are currently available at public database. Such endeavor requires a global synthesis of data, including regular auditing for consistency evaluation. Indeed, the success of barcoding approach depends on the representativeness of databases, which is only reachable by the contribution of the general scientific community. The drawback of this collaborative effort is that public databases are susceptible to inaccuracies, such as insufficient quality of the molecular data or imprecise taxonomic identifications. Here we present a semi-automatic auditing and annotation tool for reference databases to ensure the quality of the compiled data. As a proof of concept, we focused on the Canidae family and collected 935 barcodes from BOLD database with species level identification, comprising 22 species. A five-grade annotation protocol was implemented to access the concordance between taxonomic assignment and automatic clustering groups resulting from molecular data. About 90 % of the specimens were attributed to the lowest grades (either single species corresponding to more than one cluster, or single clusters comprising more than one species). Metadata curation of the specimens responsible for these inconsistences dramatically lower this figure. Among detected inconsistences, we identified one specimen labeled as Canis lupus in the cluster of the C. latrans, suggesting a misclassification of that specimen. Other inconsistences include all specimens of C. anthus, C. lycan and C. familiaris, that were grouped in the C. lupus cluster, suggesting that these specimens may all be members of the same species.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 225 P145 - SEQUENCE VARIATION OBSERVED IN 27 Y-STR MARKERS WITH U.S. POPULATION SAMPLES Becky Steffen1, Katherine Gettings1, Kevin Kiesler1, Lisa Borsuk1, Peter Vallone1

1 National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg, USA

Sequencing short tandem repeat (STR) markers on the Y chromosome allows for characterization of repeat motif variations within the Y-STR marker that cannot be determined by length- based fragment analysis with capillary electrophoresis (CE). Two commercial sequence-based assays have been run at the U.S. National Institute of Standards and Technology (NIST) with U.S. population samples (Caucasian, Hispanic, African American and Asian). The ForenSeq DNA Signature Prep Kit [1] and prototype PowerSeq 46GY System contain a total of 27 Y-STR loci between the two kits (DYF387S1, DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS460, DYS481, DYS505, DYS522, DYS533, DYS549, DYS570, DYS576, DYS612, DYS635, DYS643, and Y-GATA-H4). Data analysis was performed using an in-house pipeline based on open source software [2]. The resulting data reveals the degree of information gained through sequencing Y-STR loci and will be illustrated per population for the 27 loci. Allele calls from corresponding CE data were compared to sequence data for concordance. Discordant allele calls were further investigated to assess underlying causes such as null alleles, imbalanced peaks in multi-copy loci, flanking region insertions/deletions (indels), copy number variations, high stutter, and artifacts. 1. Verogen, ForenSeq DNA Signature Prep Reference Guide, Document #VD2018005, Rev. A (June 2018) 2. D.H. Warshauer, J.L. King, B. Budowle, STRait Razor v2.0: The improved STR Allele Identification Tool – Razor, Forensic Science International: Genetics 14 (2015) 182–186.

226 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P146 - SEQUENCING OF FULL MITOCHONDRIAL GENOMES FOR NIST POPULATION SAMPLES Kevin Kiesler1, Kimberly Andreaggi2,3, Joseph Ring2,3, Cassie Taylor2,3, Charla Marshall2,3, Peter Vallone1

1 NIST, Applied Genetics Group, Gaithersburg MD, USA 2 Armed Forces Medical Examiner System’s Armed Forces DNA Identification Lab AFMES-AFDIL, Emerging Technology Section-AFDIL, Dover DE, USA 3 SNA International, Support, Alexandria VA, USA

The U.S. National Institute of Standards and Technology (NIST) has undertaken a population sequencing study of 662 mitochondrial genomes (mtGenomes). Samples sequenced in this study represent U.S. African American, Caucasian and Hispanic population groups. The aim of the study is to increase the availability of high-quality whole mtGenome haplotypes for haplotype frequency estimations. Whole mtGenome sequencing is enabled by Next Generation Sequencing (NGS) workflows which are simplified compared to Sanger-type sequencing, which is typically limited to the mtDNA Control Region, or portions thereof, due to the labor required to produce mtDNA sequence data. For NGS, mtGenomes are amplified by long-PCR as described by Fendt et al. [1] and sequencing libraries generated using the Kapa HyperPlus Kit. Sequencing was carried out on a MiSeq FGx with a MiSeq Reagent Kit V3–600. Data analysis was performed in CLC Genomics Workbench using the AQME [2] tool developed by the U.S. Armed Forces DNA Identification Lab (AFDIL). Assessment of haplogroup resolution for mtGenome relative to control region data will be presented. Previously characterized markers for this sample set include Y-chromosome Short Tandem Repeats (Y-STRs) and Ancestry Informative Single Nucle- otide Polymorphisms (AISNPs). Ancestry prediction from mtGenome, AISNP, and Y-STR data are presented in the context of self-reporting information, with further examination in cases of dis- agreement between marker types. References: [1] Fendt et al., Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences. BMC Genomics, 10 (2009), pp.139. [2] Sturk-Andreaggi et al., AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data. Forensic Sci Int Genet., 31 (2017), 189–197.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 227 P147 - SHEDDER STATUS – FACT OR MYTH? Candy Lee1, Jiayu Tan1, Jun Yu Lee1, Yong Sheng Lee1, Zhen Qin Aw1, Mee Hui Chew1, Nurul Insyirah Binte Ishak1, Shilen Ng1, Christopher Kiu Choong Syn1

1 Health Science Authority, Biology Division- DNA Profiling Laboratory, Singapore, Singapore

The usefulness of DNA profiling in person identification to aid solving crimes is indisputable. Traditionally, the source of DNA comes from DNA-rich biological fluids such as blood, semen and saliva. But since the discovery that DNA profiles can be obtained from fingerprints, touch DNA came into the limelight for person identification and crime investigation. It is known that different factors affect the deposition of touch DNA and one of the factor which is frequently being mentioned in courtrooms is the concept of shedder status. Studies on shedder status have been conducted by different groups and have demonstrated that there is great variation between and within individuals. This raises the question of whether the concept of shedder status concept can be substantiated by scientific data. The present study answers this question by examining DNA and allele recovery 15 minutes post hand-washing in a large-scale study involving 81 individuals. Furthermore, the data collected allowed elucidation of associations between shedder status and the biometric and demographic information collected.

228 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P148 - SKETCHING MAN’S BEST FRIEND – CANINE DNA PHENOTYPING FOR FORENSIC PURPOSES Josephin Heinrich1, Burkhard Berger1, Werner Hecht2, Andreas Hellmann3, Udo Rohleder3, Uwe Schleenbecker3, Nadja Morf4, Christopher Phillips5, Walther Parson1,6, Cordula Berger1, The CaDNAP Group

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Institute of Veterinary Pathology, Justus Liebig University Giessen, Gießen, Germany 3 Bundeskriminalamt, Kriminaltechnisches Institut, Wiesbaden, Germany 4 Institute of Forensic Medicine, University of Zurich, Zürich, Switzerland 5 Forensic Genetics Unit - Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 6 Forensic Science Program, The Pennsylvania State University, University Park - PA, USA

The analysis of non-human DNA has gained importance in forensic genetics, particularly for crime scenes that lack human DNA samples. One of the most abundant non-human species relevant to forensics is the domestic dog. Currently, forensic DNA fingerprinting using Short Tandem Repeats (STRs) is the gold standard to identify canine individuals and to link crime scene traces to the donor dog. However, forensic DNA fingerprinting loses its evidential power when no canine DNA is available for comparison. In order to overcome this limitation, we propose the development of a molecular genetic tool to predict externally visible traits from dogs and thus potentially providing novel evidentiary leads to identify the dog in question or, at least, help to limit the number of possible trace donors. We have investigated 67 candidate DNA markers with known genotype-phenotype associations that were promising for the prediction of externally visible traits in dogs. We tested these markers on several dogs and developed a panel including 43 markers that produced reliable genotypes, as determined by Sanger sequencing. These markers will be combined into a single forensic molecular genetic tool by the development of a SNP-based panel for Massively Parallel Sequencing technologies. The resulting genotypes will be used to infer the phenotypic appearance of unknown dog samples by bioinformatic prediction models that will be developed specifically for the purpose. The canine phenotyping tool will be validated according to established forensic guidelines in order to allow its use in routine analyses.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 229 P149 - SMRT SEQUENCING HELP DEEPLY DELVE MITOCHONDRIAL VARIATIONS FOR MONOZYGOTIC TWINS DISCRIMINATION Lu Chen1, Guangping Fu2, Cong Bin2, Shujin Li1

1 Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, Department of Forensic Medicine, Shijiazhuang, China 2 Hebei Medical University, Department of Forensic Medicine, Shijiazhuang, China

Mitochondrial genome (mtGenome) might be a potential marker for monozygotic twins (MZT) discrimination due to its high mutation rate and heteroplasmy. Single molecular real-time (SMRT) sequencing technology is remarkable for long reads and high throughput. In the present study, we employed SEQUEL platform (Pacific Bioscience, USA) to characterize mtGenome of 16 pairs of MZ twins from minute input gDNA (~10ng) through pooling libraries with barcode. Enormous high-concordance mapped reads were produced and covered whole mtGenome with 8000× on average. We corrected at least 10 passes of subreads from one polymerase run to generate consensus sequence (CCS). CCSs reached the quality better than QV50 were assem- bled to full-length mtGenome (mean coverage depth > 50×) referring to CRS(NC_012920.1). Based on such high accuracy of CCS sequence, we found that none of the 16 MZT pairs showed sequence variation in comparison with each other, while only 4 pairs had heteroplasmy under the conditions of 50 CCSs and 5 % heteroplasmy difference. For example, twin A contained G (20+/33- reads) and A (3+/2- reads) accounting for 91.38 % and 8.62 % respectively while twin B had just G (53+/76- reads) on nt1185. These results were conservative and quite reliable in light of the ultra-deep sequencing, but still need further validation by other methods. In brief, we introduced a long read ultra-deep sequencing strategy for mtGenome from minute gDNA. Meanwhile, we found some rare mutations between MZT that brought a new sight for forensic MZT discrimination. Funding: This work was supported by the National Natural Science Foundation of China [81373246, 81671875]; and Hebei Natural Science Foundation [H2017206351].

230 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P150 - SPECIFIC m(i)RNA PROFILING FROM DNA ELUATES FOR BODY FLUID IDENTIFICATION Laiq-Jan Saidi1, Linda Helena Müller1, Burkhard Madea1, Melanie Grabmüller1

1 Institute of Legal Medicine, University of Bonn, Bonn, Germany

Knowing the cellular origin of biological stains found at a crime scene can be of significant importance for the reconstruction of this event. Conventional chemical detection methods are not very specific which is why they often give false-positive or false-negative results. In addition, only one cell type can be detected which makes these methods almost unsuitable for the investigation of minimal mixed traces. Technical and methodological developments have enabled other methods for the identification of body fluids based on differential expressed ribonucleic acids (RNA) - especially messenger RNAs (mRNAs) or microRNAs (miRNAs) - in different cell types. We compared four DNA extraction kits (PrepFiler Forensic DNA Extraction Kit, QIAamp® DNA Mini Kit, QIAamp® DNA Investigator Kit and DNA IQ™ System) to assess their relative effectiveness of the simultaneous extraction of m(i)RNA molecules and their compatibility for the prediction of body fluid identification. The evaluation included apart from the measurement of the quantity of isolated mRNA or miRNA from forensically relevant trace materials, such as dried and aged blood, liquid saliva, semen samples, vaginal secretion and mock samples, the detection of the body fluids via quantitative real time polymerase chain reaction (for miRNA) and capillary electrophoresis (for mRNA). The results showed that not all of the investigated extraction methods elute enough m(i)RNA molecules to perform an accurate body fluid identification. However, it was possible to detect specific body fluids and/or to differentiate them from each other (e.g. blood and semen) for the extraction kits which yielded enough m(i)RNA molecules.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 231 P151 - SPONTANEOUS DETERMINATION OF THE TYPE AND THE TIME SINCE DEPOSITION OF A BIOLOGICAL STAIN Chih-Wen Su1, Kuo-Lan Liu2, Hsing-Mei Hsieh3

1 Criminal Investigation Bureau, Forensic Biology Division, Taipei, Taiwan Province of China 2 Criminal Investigation Bureau, Forensic Examination Division, Taipei, Taiwan Province of China 3 Central Police University, Department of Forensic Science, Taoyuan, Taiwan Province of China

Confirming body fluid type is an important and the first step of DNA identification process for the crime scene investigation. It has been reported for identification of body fluids using mRNA profiling. Meanwhile, the differential rate of degradation between different mRNAs of biological evidence has been proven its potential for the age estimation. In this study, the expression of specific RNAs were quantified and compared to confirm its body fluid type and estimate its time since deposition (TSD) in one reaction. RNA was extracted from blood stains prepared in different time points (2 samples for each volunteer in day 0, 30, 60, 90, 120, 150 respectively) from 4 volunteers and reverse transcribed into cDNA immediately after the extraction. Quantification of a blood-specific mRNA (HBB) and 18S rRNA was executed using real-time PCR and the linear regression analysis was performed to evaluate the difference of their relative degradation. The results showed that the degradation rates were different and the ratio of the expression between HBB and 18S rRNA was in a linear relationship between day 0 to 60 (r2=0.95). TSD could be approximately evaluated by the ratio of the expression (R) with a regression equation (TSD = 10.714 × R + 18.891). After day 60, the correlation between the time and the ratio was no more significant. This method could be used to confirm the blood and evaluate TSD within 60 days at the same time in one step. This finding could be also applied in identification of the type and the time since deposition for the other body fluids.

232 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P152 - STABILITY COMPARISON OF RNA MARKERS IN BLOOD STAIN SAMPLES UNDER DIFFERENT CONDITIONS Zhilong Li1, Xiao Xiao1, Duo Peng1, Huan Tian1, Qian Wang1, Yu Tan1, Lin Zhang1, Weibo Liang1, Yanyun Wang2

1 Sichuan University, Department of Forensic Genetics- West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Sichuan University, Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education, Chengdu, China

MicroRNA(miRNA) was considered as a more stable makers than mRNA in forensic body fluid identification for its tiny size. However, few researches studied the stability of miRNA systematically. No evidence was provided to support the idea that miRNA was more stable than mRNA in body fluid identification. In this research, mRNA and miRNA markers of the same blood stain samples were detected using multiple PCR reaction and TaqMan probes respectively. Three groups were set in the experiment, namely natural degradation group, artificial degradation group and real case degradation group. In natural degradation group, there were five environment conditions: indoor without light, indoor with light, humid, dry and outdoor. In the artificial group, RNA was degraded by heating in 80 °hot water, ultraviolet irradiation and endonuclease to imitate RNA degradation. Blood stain samples were put under above conditions for different time periods. The real case group constituted two blood stain samples which revealed the complexity of real casework environment. The results showed both mRNA and miRNA were seriously degraded under humid environment. Natural illumination had a bigger impact on mRNA degradation than miRNA. Among five natural conditions, dry condition was the best to store samples. Thermal stability of miRNA was better than that of mRNA. After degradation for 360 days, at least one blood specific mRNA marker was detected in blood stain samples. Our research filled the research gap of miRNA stability and demonstrate miRNA was more stable than mRNA under some conditions.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 233 P153 - STORAGE OF SECOND WORLD WAR BONE SAMPLES: BONE FRAGMENTS VERSUS BONE POWDER Irena Zupanič Pajnič1, Eva Lina Friš1, Sara Grdina1, Jože Balažic1, Eva Podovšovnik2

1 University of Ljubljana, Medical Faculty, Institute of Forensic Medicine, Ljubljana, Slovenia 2 University of Primorska, Turistica, Faculty of Tourism Studies, Portorož, Slovenia

Bone samples may yield low-quality and low-quantity DNA and duplicated analyses of different genetic markers have to be performed for identification of missing persons. Mostly no DNA extract is left after analyses and efficient storage of bones is needed to ensure the stability of the sample over time for retesting using new markers and new technologies. Usually not all of the bone powder prepared in grinder is used for extraction and rest can be stored for future analyses. After molecular genetic analyses of 88 victims of WWII Konfin I mass grave in Slovenia (performed in 2009), fragments of femurs and bone powder that were left were stored at -20 °C. Some authors reported that long-term storage of powder results in the reduction of DNA preservation and its degradation (even at low temperature), explained by an increase in oxidative damage as a result of the enormous increase in exposed surface area. Consequently, grinding of bones as shortly prior to DNA extraction was recommended. The goal of our study was to explore the difference in DNA yield between bone fragment and bone powder frozen for 10 years. 57 WWII femurs were examined and DNA extracted from each of them using bone fragment (piece sampled next to the one used in 2009) and bone powder obtained in 2009, both taken out of freezer after 10 years of storage. Half gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen) and quantified with PowerQuant kit (Promega). Statistical analysis showed no significant difference in DNA yield comparing fragments of bones and bone powder stored at -20 °C for 10 years. Be- cause of time consuming powdering procedure we recommend to store not only the fragment of the bone, but obtained bone powder as well.

234 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P154 - STR PROFILE OF TOUCH DNA EXPOSED TO SEA WATER Hyunjung Joo1, Namyul Kim1, Minhye Kang2, Songho Han2, Sangyong An1, Taegyu Kim1, Jeongsu Noh1, Minkyu Choo1, Bongjin Jeikal1, Han-Seong Lee1

1 Korea Coast Guard Research Center, Forensic Science Research Team, Cheonan, Republic of Korea 2 Southern Regional Korea Coast Guard, Forensic Science Department, Busan, Republic of Korea

DNA identification had been limited only to bloodstains and human tissues, however, following the recent advances in the handling methods and analytic techniques for biological trace evidence, Touch DNA may now be used as evidence in court. The Korea Coast Guard brings to the surface and analyzes a large number of evidence through underwater scientific investigation, but unlike in the case of evidence on land, identification of Touch DNA exposed to the sea is still insufficient. This study therefore focuses on determining the presence of Touch DNA on evidence exposed to sea water and examines the possibility of utilizing such evidence. Imagi- nary evidence was prepared by having 3 people press with their thumb on 4 different materials for 1 minute using consistent pressure and then submerging the evidence into the ocean with the salinity of 30–33 ‰ and temperature of 9.5–16.5 °C. The evidence was collected at intervals of 24 hours and DNA sampling, quantification and STR analysis was performed. The test results have shown that STR can be reliably analyzed for up to 3 days from all types of material and that STR information allowing identification of an individual can be collected for up to 10 days. In a case when a fingerprint was left using a finger stained with another person’s blood, DNA of both the blood donor and the person who left the fingerprint were detected. This study has proven that DNA may be present even on surfaces exposed to sea water and that STR analysis is possible. To ensure marine safety, the Korea Coast Guard Research Center will continue to develop testing methods allowing exclusion of interference and reliable collection of information from evidence in the ocean that enable high level of identification.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 235 P155 - STUDY OF BIOMARKERS RELATED TO SUDDEN ARRHYTHMIC DEATH SYNDROME Tamara Kleinbielen1, María del Pilar Hernández-Sierra1, Endika Prieto-Fernández1, Félix Olasagasti2, Rubén Sevillano3, Marian M. De Pancorbo1

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 University of the Basque Country, Biochemistry and Molecular Biology, Vitoria-Gasteiz, Spain 3 Basque Institute of Legal Medicine, Pathology Service - Gipuzkoa, Donostia, Spain

Recent studies report that around 5–10 % of the autopsy cases remain unexplained in Europe, suggesting a cardiac death without heart structural alterations. In infancy population, the incidence of sudden unexpected deaths without conclusive diagnoses after autopsy range between 40 and 80 %. Almost 85 % of all sudden deaths are of cardiac origin (sudden cardiac death, SCD). SCD is usually defined as a natural, unexpected fatal event occurring within one hour from the onset of symptoms in an apparently healthy subject. Pathologists are responsible for determining the precise cause of sudden death, but this is a challenge task where molecular genetics could contribute to the comprehensive medicolegal investigation in SCD cases. SCD may occur in a structurally healthy heart and is typically caused by rare hereditary electrical disorders or arrhythmogenic cardiomyopathies. This type of SCD is labeled as sudden arrhythmic death syndrome (SADS). About one-third of deaths by SADS are due to ventricular arrhythmias, being the most common the long QT syndrome, Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). Recent genome-wide association studies identified genetic variation in NOS1AP, which encodes a nitric oxide synthase adaptor protein, as a contributor to QT interval variation. Taking this into account, the objective of this study was to evaluate five single nucleotide polymorphisms (SNPs) of NOS1AP gene, by high resolution melting (HRM), to determine their potential as diagnostic markers, as well as predictive biomarkers of the SADS.

236 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P156 - STUDY OF Y CHROMOSOME MARKERS WITH FORENSIC RELEVANCE IN LISBON IMMIGRANTS FROM AFRICAN COUNTRIES – ALLELIC VARIANTS STUDY Ângela Dente1, António Amorim1,2, Heloísa Afonso Costa2,3, Maria João Porto2, Eugénia Cunha4,5, Francisco Corte Real4,6, Cláudia Vieira Da Silva2

1 Universidade de Lisboa, Faculdade de Ciências, Lisboa, Portugal 2 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Serviço de Genética e Biologia Forenses, Lisboa, Portugal 3 Universidade da Beira Interior, Faculdade de Ciências da Saúde, Covilhã, Portugal 4 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Instituto Nacional de Medicina Legal e Ciências Forenses, Lisboa, Portugal 5 Universidade de Coimbra, Faculdade de Ciências e Tecnologia, Coimbra, Portugal 6 Universidade de Coimbra, Faculdade de Medicina, Coimbra, Portugal

Y-STR analysis is useful in the detection of DNA mixtures, particularly in cases where there is an abundance of female DNA in comparison to male DNA. Given the haploid nature of the Y chromosome, the presence of multiple peaks per marker in electropherograms may be interpreted as a mixture of male DNA. However, there have been reported cases of Y-STR marker duplications that may lead to incorrect interpretations, reflecting a need for further studies of more commonly found duplications to avoid these situations. Here, we studied 391 blood samples from male immigrants of African descent (Cabo Verde, Angola, São Tomé and Guiné-Bissau) residing in Portugal, that were involved in forensic investigations prior to 2017 at INMLCF-Delegação do Sul. Y-STR markers were co-amplified using the PowerPlex® Y23 System amplification kit (Promega) and potential marker variants were confirmed with the Y Filer™ Plus amplification kit (Applied Biosystems). Of the 391 samples studied, 38 presented marker variants, including 26 duplications, 8 microvariants and 5 null alleles. Duplications occurred in the DYS448 marker (5 %), in the DYS389 II marker paired with the DYS439 marker (0.5 %), and in the DYS389 II marker alone (0.2 %). Null alleles were observed in both the DYS448 (0.5 %) and DYS392 markers (0.8 %) and marker microvariants were found in the DYS458 (1.3 %) and DYS385 markers (0.8 %). DYS448 marker duplications were in higher frequency than has been previously reported and should then be taken into consideration whenever there are suspicions of a DNA mixture. Fur- thermore, the existence of these variants may prove useful in the identification of suspects. Seeing as it is an uncommon characteristic, the presence of these marker variants may lead to a more accurate profile match.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 237 P157 - SUDDEN UNEXPECTED DEATH IN INFANCY: NEXT GENERATION SEQUENCING ADDS VALUE TO CASES FROM CAPE TOWN, SOUTH AFRICA Laura Heathfield1, Lorna Martin1, Raj Ramesar2

1 University of Cape Town, Division of Forensic Medicine and Toxicology, Cape Town, South Africa 2 University of Cape Town, Division of Human Genetics, Cape Town, South Africa

Sudden unexpected death in infants (SUDI) is a devastating event, and unfortunately occurs frequently in South Africa. These cases are referred for a forensic post-mortem investigation to establish the cause of death; however, despite thorough analyses, some cases remain undetermined. Internationally, a molecular autopsy has assisted in resolving these types of cases by revealing genetic variants which contributed to the demise. Motivated by lack of local research in this field, a study was launched at the University of Cape Town (South Africa) in 2015 to explore the use of molecular autopsies in the medico-legal investigation of local SUDI cases. An ethical framework was established and used to prospectively recruit SUDI cases from one of the busi- est forensic facilities; Salt River Mortuary. A next generation sequencing approach was used to assess 43 genes previously associated with cardiac arrhythmias. In a particular infant, a putative pathogenic variant was identified (rs750771811 T/T) in the SCN10A gene. The variant is rare, but was homozygous in this infant, and appears to be the first time it has been observed in a SUDI victim. Previous functional studies on the amino acid residue suggested that this variant may reduce SCN5A activity, the latter which has been linked to Brugada syndrome. A genetic counselling session was arranged with the parents; a full family history was obtained, which revealed that the parents had a previous miscarriage and had recently had a second SUDI. The parents have subsequently been enrolled in the study for genetic screening and have been referred for electrocardiogram assessments. The findings highlight a new possible candidate variant to assess in SUDI cases, and also demonstrate the value of molecular autopsies to families.

238 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P158 - SUITABILITY OF CEREBRAL MATTER FOR THE FORENSIC IDENTIFICATION OF HIGHLY DECOMPOSED BODIES Katharina Helm1, Bettina Dunkelmann1, Stefan Pittner1, Christian Staufer1, Gabriele Kreindl1, Tamara Kastinger1, Eva Müller1, Waltraud Zahrer1, Ines Grießner1, Jan Cemper-Kiesslich1, Fabio Monticelli1, Neuhuber Franz1

1 Institute of Forensic Medicine, Institute of Forensic Medicine, Salzburg, Austria

Context: When bodies are highly decomposed and identification based on morphologic features is no longer possible, short tandem repeat (STR) genotyping has been proved as a convenient and reliable alternative. But at very advanced decomposition stages postmortal tissue putrefaction processes can have an effect on microsatellite instability and therefore not every type of post mortem tissue is still suitable for STR genotyping and subsequent forensic identification. In many cases bone or dental material is used but here reprocessing is very elaborate. Thus, multiple studies have focused on the eligibility of soft tissue and organs where DNA extraction is a lot easier and faster. Aims: By evaluation of different cases in which a successful forensic identification was only possible with DNA material extracted from specific tissues we aim to compare differential suitability of soft tissues for DNA analysis of highly decomposed bodies. Material and Methods: Systematic classification of decomposed bodies and analysed organs to evaluate the qualification of soft tissue for DNA profiling in respect to the degree of decomposition. Conclusion: We detected differences in the suitability of various soft tissues for DNA-based identification. Cerebral matter turned out to be one of the most useful samples in highly decomposed bodies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 239 P159 - TAPHONOMIC EFFECTS ON DNA DEGRADATION IN POST-MORTEM BODY TISSUES IN AN AUSTRALIAN SETTING Samara Garrett-Rickman1, Maiken Ueland1, Dennis McNevin1, Shari Forbes2

1 University of Technology Sydney, Centre for Foresic Science, Sydney, Australia 2 Université du Québec à Trois-Rivières, Département de chimie- biochimie et physique, Trois-Rivières, Canada

Traditional post mortem interval (PMI) estimation techniques rely on subjective assessment and calculations based on analysis through entomology, thanatochemistry and visual observations (total body scoring). Although estimation is possible, the window of PMI is often large and inconsistent, and many techniques have been shown to be relevant to specific climates. Thus, research into more accurate techniques and understanding of the underlying processes is needed but has been limited due to ethical and practical constraints. However, through taphonomic facilities, it is now possible to observe human decomposition. This study investigates taphonomic effects on the recovery and degradation rate of nuclear DNA (nDNA) from post-mortem human tissues after being exposed to the natural environment. The degradation of nDNA was determined using real time-qPCR with both a short and long amplicon to create a degradation index. Variables influencing degradation included: temperature, accumulated degree days, humidity, rainfall, and scavenger activity. The goal of this presentation is to highlight the variability in taphonomic effects on DNA within the Australian environment and update the forensic science community on the research being conducted at the Australian Facility for Taphonomic Experimental Research. The forensic science community will benefit from a better understanding of the post-mortem processes on the availability of genetic material as well as a new potential method for the estimation of post-mortem interval.

240 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P160 - THE ANALYSIS OF ANCIENT DNA FOR FORENSIC APPLICATIONS Sun Hee Park1, Hyo Sook Kim2, Eun Hye Kim3, Man Il Kim1, Hye Hyeon Lee1, Ki Yoon Eom1, Eun Jung Lee1, Pil-Won Kang2, Yang Han Lee1, Tae Sop Cho4, Chang Gyun Han4, Eung Soo Kim2

1 Forensic DNA Division, National Forensic Service Gwangju institute, Gwangju, Republic of Korea 2 Forensic DNA Division, National Forensic Service, Wonju- Gangwon-do, Republic of Korea 3 Forensic DNA Division, National Forensic Service Seoul institute, Seoul, Republic of Korea 4 Department of History, Yonsei University, Seoul, Republic of Korea

Among the evidence in criminal cases assigned to the National Forensic Service is an old bone that has long been buried in the ground. Also, in many cases, an old bone may be the only clue in a criminal case. By separating DNA from old bones and identifying it, one can classify the remains as belonging to a missing person or a victim of any crime. Therefore, while it is not easy to separate identifiable DNA from old bones, doing so is very important. Bronze Age bones have been excavated from the Jeong-Seon Medun Cave in South Korea’s Gang-Won Province, and it would also be very useful in forensic science to separate DNA from the bones and analyze the data to obtain meaningful results. We used various research methods such as analysis of short tandem repeat and mitochondrial DNA, which are used in forensic genetics, to separate DNA from the Bronze Age bones, and herein we presented our results. In addition, both the excavation participants and the researchers tested the results in the same way to check for contamination. Furthermore, the experiment was conducted under experimental conditions suitable for ancient DNA (DNA separated from ancient specimens), such as the separation of time and space, referring to the existing experimental method that deals with the ancient DNA. Separating the ancient DNA from ancient specimens has proved difficult in many ways, but applying this result to forensic science may help further advance forensic techniques and obtain good results from old bones, which often provide the only clues available.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 241 P161 - THE BREED MAKES THE DIFFERENCE: PEDIGREE COMPOSITION, CANINE STR ALLELE FREQUENCIES, AND RANDOM MATCH PROBABILITIES Burkhard Berger1, Cordula Berger1, Josephin Heinrich1, Werner Hecht2, Andreas Hellmann3, Uwe Schleenbecker3, Udo Rohleder3, Nadja Morf4, Walther Parson1,5 The CaDNAP Group

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Institute of Veterinary Pathology, Justus-Liebig-University Giessen, Giessen, Germany 3 Kriminaltechnisches Institut, Bundeskriminalamt, Wiesbaden, Germany 4 Institute of Forensic Medicine, University of Zurich, Zürich, Switzerland 5 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

Domestic dogs are popular and as integral part in everyday life they can become relevant to forensic DNA analysis, which is typically conducted by canine STR analysis followed by assessing the weight of evidence of a matching DNA profile, e.g. by random match probability (RMP) calculations. Noteworthy in this context is the outstanding diversity of dogs. Breeders have exerted selective pressure throughout a long period for a variety of purposes such as hunting, tracking, or herding, creating an extraordinary variety of modern dog breeds, unparalleled in any other animal species. This diversity applies also to forensically used STR markers as will be exemplarily shown by STRUCTURE and principal component analysis demonstrating that STR characteristics largely correspond with the breed affiliations of the underlying dog individuals. This challenges the determination of a suitable population sample that provides a reliable basis for computing allele frequencies and correction factors (Theta, FST) applicable for conservative RMP calculations. As a practicable approach we propose to approximate the breed composition of the population sample as closely as possible to that of the actual dog population, which would mean that popular breeds such as German Shepherds or Labrador Retrievers constitute a large part, whereas rare breeds are represented by one or only few individuals. To test the robustness of this suggested approach, a data set of 13 STR genotypes derived from nearly 1,200 dogs and representing more than 166 breeds was used to create population sample subsets with varying breed compositions. The impact of the different breed compositions on resulting RMP-values for members of popular and rare breeds will be discussed.

242 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P162 - THE EFFECT OF CLIMATIC SIMULATIONS ON DNA PERSISTENCE ON GLASS, COTTON AND POLYESTER Carol Chen1,2, Alice Pistono1,3,4, Suzanna Ryan5, Bianca Szkuta6,7, Georgina Meakin1,2

1 Centre for the Forensic Sciences, University College London, London, United Kingdom 2 Department of Security and Crime Science, University College London, London, United Kingdom 3 Division of Biosciences, University College London, London, United Kingdom 4 Department of Natural Sciences, University College London, London, United Kingdom 5 Pure Gold Forensics, Pure Gold Forensics, Redlands, USA 6 Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Macleod, Australia 7 School of Life and Environmental Sciences, Deakin University, Melbourne, Australia

It is important to understand the variables impacting DNA persistence when considering the recovery and evaluative interpretation of DNA from crime scenes. Whilst it is known that temperature, humidity and UV affect DNA persistence, little research has been conducted to explore these effects in a combined manner. We present two studies in which a climate chamber was used to simulate climatic conditions over a repeating 24-hr period. Aliquots of ~50ng DNA were added to each substrate and DNA recovered at 0, 1, 3 and 7 days after deposition. Samples were run in triplicate and extracted, quantified and profiled. The first study investigated the effect of typical Southern English winter (1–6 °C; 80 % av. humidity; 8hr sunlight) and summer (15–26 °C; 64 % av. humidity; 14hr sunlight) days on the persistence of DNA on glass and cotton. DNA was recovered using wet and dry swabs from glass and mini-tapes from cotton. The second study investigated the effect of typical Northern Italian winter (0–18 °C; 70 % av. humidity; 12 hr sunlight) and summer (13–35 °C; 60 % av. humidity; 16 hr sunlight) days on the persistence of DNA on cotton and polyester. DNA was recovered using wet and moist swabs from both fabrics, as an additional experiment showed this method to have a better recovery efficiency from cotton than mini-taping (24 % vs 2 % recovery, respectively). Quantities of DNA on all substrates significantly declined over 7 days under summer conditions (p<0.05), and significantly more DNA persisted on cotton in both studies under conditions of winter than summer (p<0.01). These results contribute to our understanding of DNA persistence under different climatic conditions and will help inform investigators’ DNA recovery strategies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 243 P163 - THE EFFECT OF HYDROLYSIS ON DNA Kristina Schulze Johann1, Heidi Pfeiffer1, Marielle Vennemann1

1 University Münster, Institute of legal medicine, Münster, Germany

The aim of this preliminary study was to detect the level of DNA hydrolysis in aged blood and saliva samples. DNA hydrolysis can lead to deamination of cytosines. By treating the hydrolysed DNA with AmpErase™ Uracil N-Glycosylase (UNG) abasic sites should be created, making them more susceptible to DNA strand breaks induced by subsequent heat or ultrasonic treatment. Samples were stored under various conditions and DNA was extracted using DNA IQTM Case- work Pro Kit for Maxwell 16 before treatment with UNG and heat /ultrasound. DNA was then quantified using PowerQuant System (Promega), which includes a “degradation marker” to detect strand breaks. Provided DNA hydrolysis is time-dependent during sample storage, comparing the degradation level of the samples treated with UNG and the same samples not treated with UNG should correlate to the time since deposition. Initial results will be discussed.

244 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P164 - THE EFFECT OF THE HIGH QUANTITY OF FEMALE DNA EXTRACTED FROM VAGINAL FLUID ON THE DETECTION OF Y-STR PROFILE AND THE QUANTITATIVE VALUE OF MALE DNA Hyun Kuk Cho1, Jin Myeong Lee1, Jin-Young Kim1, Sung Kyoung Choi1, Dong Ho Choi1, Sang‑ok Moon1

1 National Forensic Service, Division of DNA Analysis, Seoul, Republic of Korea

It is the key to detect male genotype from vaginal fluid in sexual assault cases. However, it is difficult to obtain the autosomal short tandem repeats (STRs) profile of a male in a high female DNA background. Therefore, it is important to obtain the Y chromosome STR profile in the cases. To investigate the effect of high quantity of female DNA extracted from vaginal fluid on the detection of Y-STR profile, two Y-STR kits (PowerPlex® Y23 amplification system and YfilerTM Plus PCR amplification kit) and DNA Control 007 were used. The limit of detection (LOD) of PowerPlex® Y23 and YfilerTM Plus for the obtaining of Y-STR full profile was 0.05 and 0.1 ng in this system. It was tried to detect Y-STR profile on the samples containing the various levels of female DNA and the fixed level of male DNA. The results showed that there was no significant difference on the detection of Y-STR profile in the samples. In sexual assault cases, there were many cases that the detection of Y-STR full profile was available on forensic samples containing the male DNA less than the LOD. At this point, we wondered whether the quantitative value of male DNA was affected by the high level of female DNA. Using the Quantifiler Trio DNA Quantification Kit, the quantitative values of the male DNA were measured on the samples containing the various levels of female DNA and the fixed level of male DNA. The results showed that the quantitative values of male DNA were measured lower than that of control dependent on the high level of the female DNA. This study is expected to give the reliability of the tests in which Y-STR full profile is obtained using the forensic samples with the quantitative values of male DNA less than LOD or single cell.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 245 P165 - THE EFFECTS OF VARIOUS HOUSEHOLD CLEANING METHODS ON DNA PERSISTENCE ON MUGS AND KNIVES Vivienne Li1,2, Hayley Toogood2, Suzanna Ryan3, Georgina Meakin1,2

With the prevalence of forensic science in popular media, offenders are becoming more forensically aware and can employ precautionary methods, such as cleaning used items or rubbing away fingermarks, to reduce their traces left at a crime scene. We present two studies in which the effects of various cleaning methods on DNA persistence on commonly encountered casework exhibits, specifically knives and mugs, were investigated. Aliquots of DNA were added to the substrates, allowed to dry, and then the substrates were either sampled directly or were cleaned and then sampled. Samples were collected with wet and dry swabs, in triplicate, and extracted, quantified and profiled. In the first study, aliquots of 300ng DNA were added to the handles of plastic and wooden handled knives, which were then cleaned with a cloth in a sink of water, diluted washing up liquid (10ml in 1.5l water) or household bleach (36ml in 1.5l water). In the second study, aliquots of 50ng DNA were added to the rims of ceramic, glass and steel mugs, which were then cleaned with a dry or wet cloth, or by wiping with a cloth after applying a cleaning product (0.7ml washing up liquid or two sprays of household bleach spray) directly into the mug and then rinsing it with water. In both studies, DNA was not detected on items after cleaning with washing up liquid or household bleach, irrespective of the differences in amounts of DNA deposited, substrates, and cleaning methods. Even without a cleaning product, rubbing with a dry cloth significantly decreased DNA recovery from the mugs (p<0.05), regardless of the mug substrate. These results contribute to our understanding of the impact of various cleaning methods on DNA recovery at the crime scene and will help inform DNA recovery strategies.

246 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P166 - THE INFLUENCE OF THE SHEDDER STATE ON DNA TRANSFER Max Schmidt1, Peter Wiegand1, Marielle Vennemann2, Heidi Pfeiffer2

1 Institute of Legal Medicine, Forensic Genetics, Ulm, Germany 2 Institute of Legal Medicine, Forensic Molecular Biology, Münster, Germany

Various different parameters and circumstances affect the DNA transferability and accordingly the quantity of DNA left, determining the success of DNA typing. A considerable parameter affecting the DNA transfer is the so-called “shedder” state, which describes an individual’s ability to pick up and deposit DNA. To evaluate this proposition, a primary DNA transfer experiment was conducted to classify individuals into corresponding shedder groups, namely “good shedders” and “bad shedders”. Subsequently, our study aimed to assess the influence of the shedder state on secondary and tertiary DNA transfer: Can DNA of a good shedder be placed on a crime scene by a bad shedder even though the good shedder is not connected to the crime and has not been at the crime scene? To answer this question sets of secondary and tertiary DNA transfer studies were performed. Our results show that a shedder state classification was possible for the majority of the participants and the shedder state seems to have an impact on secondary and tertiary DNA transfer. It was further shown that bad shedders might act as vectors; hence, they were able to transfer excessive DNA quantities from good shedders onto further objects. Moreover, we demonstrated that good shedders can transfer minute amounts of bad shedders’ DNA to further objects. The presence of the contributors´ DNA was verified using three different fully continuous biostatistical models (EuroForMix, Genoproof, STRmix). The Likelihood ratio concordance and deconvolution tool applicability was evaluated. The resulted data provide an estimation of the comparability among each other but also in terms of semi-continuous models.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 247 P167 - THE PRESENCE OF BACKGROUND DNA ON COMMON ENTRY POINTS TO HOMES Bianca Szkuta1,2, Jack Reither1, Xavier Conlan1, Roland van Oorschot2,3

The successful recovery of DNA traces from objects and surfaces within public and private environments is dependent on, but not limited to, the history of use, surface nature, and environmental conditions. In such settings, the prevalence and collection of ‘background’ DNA (BDNA) from the sampled surface may impact detectability of targeted person(s). As such, it is becoming increasingly relevant to understand how much BDNA is present on, and who might contribute to, samples collected from surfaces frequently targeted for DNA sampling in criminal investigations. This study investigates the presence of BDNA on common entry points in five homes occupied by known individuals. Each home had at least two occupants, and residents were over the age of 18 and not directly related to one another. Samples were collected from internal and external windowsills and edges within bedrooms and living rooms (n = 96), as well as front and rear door handles (n = 36). Questionnaires provided information relating to the history of the surfaces, including when, how and who last used and cleaned them. The amount of DNA and the types of profiles generated from door handles did not differ between front and rear locations, and internal and external environments. In contrast to door handles, less DNA was obtained from external window sills and edges compared to the equivalent internal surfaces. Majority of samples from external sites also yielded limited to no profiling information, while 3-person mixtures were most commonly generated from internal sites. These findings contribute to our current knowledge on the prevalence of BDNA and will assist scientists in making informed decisions on sampling strategy.

248 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P168 - THE SEQUENCE IDENTIFIER (SID): AN OPERATIONAL NOMENCLATURE FOR MIXED-DNA CASEWORK USING MPS Brian Young1, Tom Faris1, Abdulkareem Alali1, Luigi Armogida2

1 NicheVision Forensics- Inc., Research, Akron, USA 2 NicheVision Forensics- Inc., Management, Akron, USA

The use of MPS in casework has been hindered by the lack of a nomenclature that meets the operational needs of routine casework. The SID nomenclature offers a compact labeling system for computer analysis of mixed MPS casework data. SID is a hash value converted to a base- 26 value comprised solely of capital letters from the English alphabet. SID letters are dynamically allocated within loci allowing unique and deterministic labeling of all sequences using typically only the first 2 letters of the SID. The SID nomenclature will be demonstrated in the ArmedX- pert™ mixture analysis tool which has been modified to analyze sequence-based data. For example, an allelic fragment equivalent to STRSeq GenBank accession MK570033.1 is labeled D1S1656 15.3 JE, where the 2-letter SID is prepended with the locus name and CE-equivalent length. The N-1 LUS stutter is labeled D1S1656 15.3 JE.AC. ArmedXpert™ implements a “dot” connector to denote a parent-child relationship between the allele and the stutter artifact. If (for example) a C->T sequencing error occurs 18 nucleotides in the upstream flank, then the fragment is labeled D1S1656 20.3 JE`WE with a “tick” connector denoting a parent-child relationship between the allele and the sequence artifact. Once labeled, alleles and artifacts can be compactly displayed and efficiently filtered by ArmedXpert software. The SID along with dot and tick connectors compactly communicate the software-called categorization of sequences and can be printed in an audit trail along with the raw sequences, and other nomenclature formats as required for profile reporting and databasing.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 249 P169 - THE USING OF MASSIVELY PARALLEL SEQUENCING OF MITOCHONDRIAL DNA TO ASSIST THE MISSING PERSON IDENTIFICATION: HUMAN REMAINS IN THE WILD Sirirat Mienkerd1, Anillada Nettakul1, Worawee Waiyawuth1

1 Central Institute of Forensic Science, DNA Division, Bangkok, Thailand

On April 2016 a man was reported missing at Phu Khiao wildlife sanctuary, Chaiyaphum province, Thailand. After a year, the human remains and twenty of evidences which suspected belonging to the missing person were discovered in the wild. The DNA from twenty evidences and the occipital bone were extracted and amplified by Investigator IDplex Plus Kit and GlobalFiler™ PCR Amplification Kit. The identification of the missing person was performed by the kinship testing between the bone and the younger sister. According to the hot and humid environment in the wild, the bone specimen was highly degraded by the moisture and the soil microorganisms. The recovery of the low amount of DNA resulted the partial and incomplete STR profile. The retrieval of 5 STR loci lent to the failure in the full sibling testing. The determination of the maternal lineage by mtDNA was applied in this case. As a consequence of the DNA insuffi- ciency, the conventional sanger sequencing of the mtDNA hypervariable regions (HV1 and HV2) was shifted to the massively parallel sequencing using Ion Torrent Personal Genome Machine (PGM). With the potential and a high sensitivity technique, the mtDNA haplotype was acquired from the degraded bone and matched against the younger sister. The result showed the confirmation of the same maternal lineage. The application of the mtDNA and the massively parallel sequencing technique assisted the identification of missing person from the degraded bone which exposed to the inappropriate environment for a long time. Keyword: Missing person, Mitochondrial DNA, Massive parallel sequencing.

250 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P170 - THE VAMPIRE PROJECT Andrzej Ossowski1, Maria Szargut1, Joanna Arciszewska1, Sandra Cytacka1, Mirosław Parafiniuk2, Grażyna Zielińska1, Grzegorz Machalski3, Mariusz Holicki3, Grzegorz Kurka4, Joanna Drath1

1 Pomeranian Medical University in Szczecin, Department of Forensic Genetics, Szczecin, Poland 2 Pomeranian Medical University in Szczecin, Department of Forensic Medicine, Szczecin, Poland 3 West Pomeranian Oncology Center, West Pomeranian Oncology Center, Szczecin, Poland 4 The Museum of History of Kamień Pomorski, Kamien Pomorski, Poland

In 2014 in a small city in Pomerania, Kamien Pomorski archeologists found strange burial. The skeleton had strange damage to the femur and tibia bones. The next shocking discovery was a brick placed in the mouth of the deceased. The person found was buried on the edge of the cemetery, some distance from the other graves. the burial was called the vampire’s burial, and the information about this burial went around the world. The information was reported by such websites as CNN, Daily Mail, The Sun, National Geographic, etc. The anthropological and archaeological analysis carried out showed that the remains belonged to a man, dark-eyed, dark- haired, from a different population than residing in Pomerania. In the media, it was written that the appearance of this man was supposed to provoke fear. Was it really like that? The vampire remains came to the Museum in Kamien Pomorski. After 3 years, the museum turned to the Po- meranian Medical University with a request for further research, the results of comprehensive studies have been surprising.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 251 P171 - TIGRIS ID: SCIENCE AGAINST AN ILLEGAL TRADE OF PANTHERA TIGRIS IN THE HEART OF EUROPE Daniel Vanek1,2,3, Lenka Vankova1, Pavla Rihova4, Eva Tikalova1, Edvard Ehler1, Simona Dalihodova1, Jitka Votrubova1

1 Forensic DNA Service, DNA laboratory, Prague, Czech Republic 2 Charles University in Prague, 2nd Faculty of Medicine, Prague, Czech Republic 3 Institute of Legal Medicine, Bulovka Hospital, DNA laboratory, Prague, Czech Republic 4 Czech Environmental Inspectorate, Cites, Prague, Czech Republic

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Some recent textbooks of TCM still recommend formulas containing various animal tissues such as tiger bones, antelope, buffalo or rhino horns, bear or snake bile. In June 2018, Czech Republic authorities raided premises in Prague and other locations, revealing a tiger slaughterhouse at the center of an international criminal trade ring. The raids, under the name Operation Trophy, were the culmination of two-and-a-half years of work and employed more than 200 enforcement officers from customs, police and the Czech Environmental Inspectorate. In the illegal slaughterhouse, they found a freshly killed tiger, shot through the eye to leave its skin undamaged, a boiler for preparation of tiger glue, many tiger claws, bones and skins, and dozens of dead animals, often in a state of decay. DNA analyses were focused on specie identification and individual identification of Panthera tigris samples. Individual identification of Panthera tigris samples was accomplished thanks to the research activities under the research grant of Czech Ministry of Interior. A summary of the DNA typing results for this case, as well as data from the development and validation, will be presented.

252 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P172 - TIME ESTIMATION OF FOUR DIFFERENT SURFACES EXPOSED IN MARINE ENVIRONMENTS USING MARINE BIOFOULING Han Seung Lee1, Sang Mok Jung2, Seong Hee Han2, Chang Yeon Lee2, Won Bin Jeon2, Ga Yeon Kim2, Tae Soo Kim2, Bongjin Jeikal1, Hyun Woung Shin2

1 Korea coast guard research center, Forensic science research team, Cheonan-si Chungcheongnam-do, Republic of Korea 2 Soonchunhyang University, Department of Life Science and Biotechnology, Asan-si Chungcheongnam-do, Republic of Korea

Marine biofouling is the accumulation of microorganisms, plants, algae or animals on man-made structures on surfaces such as plastics, iron, stainless, clothes, wood and others in seawater. There are several stages of biofouling, such as organic materials, microorganisms, seaweeds and animals. This is not only the stages, but it is also strongly related to times such as seconds, minutes, hours, weeks, months, and years. Therefore, this study analyzed the time estimation relevance using biofouling process. In this experimental design, four different surface panels (10×10 cm2) were immersed in sea area at a depth of 1 m and 4 m, respectively. Every month, the biofouling was identified and the new panels were immersed. Based on ASTM methods, the fouling organisms were measured for their attachment rate, length and individuals. For that, the fouling rate was followed by pvc, sus, cloth and wood. After three months, those four surfaces were covered biofouling over 90 %. The dominant species were Balanus sp. Encrusting bryozoans, Spirorbis sp. Hydroides sp. Ulva sp. and Crassostrea sp.. However, Balanus sp. sequentially observed every month. Animal fouling were higher attachment than plants. The fouling was related to temperatures, for example, the summer season was higher fouling organisms. This study can be used to estimate marine dumping time for accidents, bodies and crime tools in the sea areas.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 253 P173 - TIME SINCE DEPOSITION OF BIOLOGICAL FLUIDS USING RNA DEGRADATION Thomas Bird1, Graham Williams2, Laura Walton-Williams3

1 Staffordshire University, PhD Researcher, Stoke-on-Trent, United Kingdom 2 Staffordshire University, Head of Department - Criminal Justice and Forensics, Stoke-on-Trent, United Kingdom 3 Staffordshire University, Associate Dean - Recruitment, Stoke-on-Trent, United Kingdom

In forensic cases where intercourse has taken place, bodily fluids are usually left behind. Bodily fluid identification (BFID) of the type and origin can provide important information regarding what occurred and who, however BFID struggles to determine time since deposition (TSD) of biological stains. According to the Crime Survey in England and Wales (CSEW) in 2017, it shows that the majority of sexual offences that occur are committed by people already known to the victims, roughly around 90 %, with 56 % of these being committed by partners or ex partners. In these cases, the defence is that their DNA will be present because they are in a relationship or were in a prior relationship. To combat this defence, looking into the degradation of RNA in biological stains like semen can potentially provide a timeline of the time since a stain was deposited. As no two crime scenes are identical, external factors may play a part in the rate of degradation of RNA such as temperature and liquid or stain. In this research a number of semen specific mRNA and miRNA genes alongside housekeeping genes were analysed as to the degradation levels over a period of time. The mRNA genes chosen based on various literature were tissue specific sperm genes; Protamine 1 and 2 and tissue specific seminal fluid genes; Kallikrein 3 and Transglutamine 4 with the reference mRNA gene Glyceraldehyde 3-phosphate dehydro- genase (GAPDH) used as a control. These semen stains were extracted periodically and quantified using qPCR generating real-time levels of RNA which can then be compared to the levels of fresh semen. The findings of this research will be discussed in this paper. This research can then be compared to chemical TSD papers to determine whether a chemical or a biological route is more accurate.

254 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P174 - TOWARDS SUCCESSFUL IDENTIFICATION OF CHILD‑VICTIMS FROM SEVERAL DECADES AGO Sandra Cytacka1, Joanna Arciszewska1, Maria Szargut1, Grażyna Zielińska1, Katarzyna Jałowińska1, Andrzej Ossowski1

1 Pomeranian Medical University, Department of Forensic Genetics, Szczecin, Poland

Human identification has been a challenge for forensic geneticists for years, especially when the research material are bones with a high degree of DNA degradation. Through many years of research as part of the project of the Polish Genetic Database of Victims of Totalitarianism www. pbgot.pl (PBGOT), scientists from the Pomeranian Medical University in Szczecin gained extensive experience in analysis of degraded bone material. The project was created 7 years ago, and since that time specialists from PBGOT’s team examined hundreds of human remains, among them those of men, women and children. In the case of adults, fragments of femurs or teeth are usually collected for the examination, results obtained from these bone fragments show a fairly high concentration of DNA. During the research, the scientists noticed that the routine approach doesn’t work apply to examining the remains of children. In order to obtain more satisfactory results, DNA was extracted from various parts of the skeleton and significantly better results were obtained from the petrus bone. The research material was collected from 40 children’s remains exhumed at the detention center in Białystok and contained also another available bone fragment from each individual. The comparison of the obtained results within DNA concentration, the number of amplified STR and Y-STR markers shows that in the case of bone material with a high degree of DNA degradation coming from children, the best results are obtained from the petrus bone.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 255 P175 - TWO LOCI ‘EXCLUSION’ OF TRUE PATERNITY IS DUE TO GENETIC DISORDER IN A CHILD Sergei Borovko1, Alena Shyla1, Arthur Luhauniou1, Siarhei Aranovich1, Yulia Zayerka1

1 State Forensic Examination Committee, Forensic biology and genetics, Minsk, Belarus

We describe a paternity case with maternal uniparental disomy of chromosome 2 in a three- year-old boy. The proband has a diagnosis of Miller-Dieker syndrome, although this syndrome has not been confirmed by FISH analysis with a specific LS1 LIS1 Miller-Dieker probe 17p13.3 (Vy- sis, Abbott Laboratories). Magnetic resonance imaging of the brain determined the presence of cortical dysplasia with lissencephaly. Chromosome analysis showed a normal 46, XY karyotype. STR analysis with the use of GlobalFiler and Verifiler Plus Kits (Applied Biosystems), PowerPlex Fusion 6C System (Promega) revealed the absence of paternal alleles and presence of two maternal alleles at D2S441, D2S1338 loci in the proband. The origin of the proband’s TPOX alleles was not possible to determine using microsatellite analysis. The rest 22 STR markers were all fully informative and served to confirm paternity. In addition, Y-STRs were analyzed using Yfiler Kit (Applied Biosystems) and the same haplotype was determined in the child and his putative father. Genetic profiles of autosomal STRs, Y-STRs and amelogenin for DNA isolated from the child’s buccal swabs and blood were the same. With massive parallel sequencing on HID Ion GeneStudio S5 System using Precision ID Global- Filer NGS STR Panel v2 (Applied Biosystems) we confirmed the presence of two alleles of maternal origin at D2S441, D2S1338 loci and identified two maternal alleles at additional locus D2S1776 located on chromosome 2 in the proband. Identification of maternal and paternal isoalleles at D7S820 and D8S1179 gave us strong evidence to prove paternity. Further genetic counselling for the proband with the use of genetic data revealed in paternity test is recommended.

256 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P176 - UNDERSTANDING THE BEHAVIOR OF STUTTER THROUGH THE SEQUENCING OF STR ALLELES Sarah Riman1, Hariharan Iyer2, Lisa Borsuk1, Peter Vallone1

1 U.S. National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg, USA 2 U.S. National Institute of Standards and Technology, Statistical Design, Analysis and Modeling Group, Gaithersburg, USA

This work explores the influence of several variables on stutter formation across sequenced autosomal STR loci (simple, compound, and complex motifs) and different alleles within each locus. The variables are sequence variations within the repeating motifs and flanking region [1, 2]; longest uninterrupted repeat sequence (LUS) [3]; parental allele length [3]; and base pair content and length value of each repeating motif from which the stutter has generated [3, 4]. Over six hundred unrelated individuals from different populations were amplified with the prototype PowerSeq 46GY System and sequenced on the Illumina MiSeq platform. Raw FASTQ files were analyzed with STRait Razor v3 [5]. Stutter ratio was calculated for motifs that exhibited stutter using the ratio of the observed coverage of the stutter sequence at (N-1) position to the observed coverage of the allelic sequence. Understanding the mechanism by which stutter is formed will help in establishing probabilistic models for the prediction of stutter rate and interpretation of sequence-based STR alleles. 1. Woerner A.E. et al. Flanking Variation Influences Rates of Stutter in Simple Repeats, Genes (Basel) 8 (2017) 1–20. 2. Woerner A.E. et al. Compound stutter in D2S1338 and D12S391, Forensic science international. Genetics 39 (2019) 50–56. 3. Brookes C. et al. Characterising stutter in forensic STR multiplexes, Forensic science international. Genetics 6 (2012) 58–63. 4. Vilsen S.B. et al. Stutter analysis of complex STR MPS data, Forensic science international. Ge- netics 35 (2018) 107–112. 5. Woerner A.E. et al. Fast STR allele identification with STRait Razor 3.0, Forensic science international. Genetics 30 (2017) 18–23.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 257 P177 - UPDATE IN THE GENETIC IDENTIFICATION OF SKELETAL REMAINS FROM VICTIMS OF THE SPANISH CIVIL WAR AND POSTERIOR DICTATORSHIP Miriam Baeta1, Carolina Núñez1, Caterina Raffone1,2, Eva Granizo1, Leire Palencia-Madrid1, Sergio Cardoso1, Lourdes Herrasti2, Francisco Etxeberria2,3, Marian M. De Pancorbo1

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 Society of Sciences Aranzadi, Physical Anthropology, Donostia, Spain 3 University of the Basque Country, Legal and Forensic Medicine, Donostia, Spain

In this study, we present our experience in the genetic identification of skeletal remains recovered from graves of the Spanish Civil War (1936–1939) and posterior dictatorship (until 1970s). As result of that period of conflict, it is estimated than around 114,000 victims disappeared along the Spanish territory. Unfortunately, 80 years after the conflict broke out, the percentage of victims recovered or identified still represents a small portion of the totality. Approximately 9,000 victims have been recovered from around 700 mass graves (out of the ~2,000 graves estimated) during the last eighteen years. Furthermore, due to the long time elapsed since the end of the conflict and the challenges associated to old skeleton samples, conventional methods may not be sufficiently discriminative to identify the unknown person. In this context, genetic analyses offer an efficient tool to unveil the victim identity. Up to now, we have carried out the genetic analysis of more than 500 human remains from graves located in the Spanish territory, particularly from the northern half. Autosomal STRs, Y-STRs, X-STRs and/or mtDNA were studied in order to establish kinship relationship with presumptive relatives and reach a successful identification of the unknown remains. In the last years, our efforts have focused on overcoming the limitations of this kind of analysis: limited quality and quantity of DNA recovered from the remains, partial genetic profiles from these samples and scarce number of appropriate family members for genetic comparisons. With this aim, we have optimized the process from the DNA extraction to the matching search -an increased number of relatives’ profiles in the genetic databases (n>800) - in order to raise the number of identifications.

258 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P178 - USE OF FORENSEQ™ DNA SIGNATURE PREP KIT ON IDENTIFICATION OF FORMALIN-FIXED TUMOR TISSUES Lihong Fu1, Peipei Deng1, Qingqing Du1, Bin Cong1, Shujin Li1

1 Hebei Medical University, Department of Forensic Medicine, Shijiazhuang, China

This study evaluates the application of massive parallel sequencing (MPS) in degraded tumor tissues for purpose of body source identification. ForenSeqTM DNA Signature Prep kit was used to detect 27 autosomal STRs, 24 Y-STRs, 7 X-STRs and 94 identity informative SNPs of the 12 paired degraded tumor/normal tissue DNA. The results indicated Miseq FGx sequence platform can detect the intra-STR sequence variants, and supplement dropped-out allele by CE, and exhibit more sensitive to low ratio STR and SNP alleles based on massive sequence data, which can offer more genetic information in order to improve the likelihood ratio of identification with tumor samples. However, the high mutation rate of STR in tumor tissue makes its benefit of isoalleles discounted. On the contrary, besides a shorter amplicon size of SNP (less than 160bp) in ForenSeqTM DNA Signature Prep Kit, up to 94 iiSNPs simultaneous typed in one panel, which produced LR up to 1039. These results highlight that increased discrimination power for tumor tissues can be obtained by MPS. In addition, heterozygote of tumor tissue is more imbalanced than that of normal tissue, and sufficient read depth, analytical and interpretation threshold for each locus are needed to reset in laboratory. ForenSeqTM DNA Signature Prep Kit can be used as a valid tool for the individual identification of degraded tumor tissues.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 259 P179 - VALIDATION AND ASSESSMENT OF AN OPTIMIZED MASSIVELY PARALLEL SEQUENCING WORKFLOW FOR WHOLE MITOCHONDRIAL GENOMES OF REFERENCE SAMPLES Sara Rožić1, Viktorija Sukser1, Marina Korolija1, Lucija Barbarić1

1 Forensic Science Centre “Ivan Vučetić”, Biology and Fibers Department, Zagreb, Croatia

Massively parallel sequencing (MPS) technology shaped the era of forensic genomics, particularly regarding mitochondrial DNA (mtDNA), one of the most valuable markers in cases when conventional nuclear DNA typing fails. The technology enabled higher throughput and greater sensitivity, which comes with a greater possibility of errors. In order to produce reliable forensic results, validation is the first step when incorporating new methodology into routine casework. The work presented here is part of our ongoing internal validation of MPS workflow for sequencing whole mtDNA from reference samples using Nextera® XT (Illumina®) protocol on MiSeq FGx™ instrument. Through studies of sensitivity, mixtures, repeatability and reproducibility, as well as concordance and contamination studies, we aimed to ensure accuracy and reliability of generated data from manually prepared libraries. To cover the entire mitochondrial genome, reference samples were enriched by long range PCR, resulting in two overlapping fragments of length 9.1 kb and 11.2 kb. Three different DNA polymerases were tested for optimal conditions, prior to library preparation step. Repeatability and reproducibility were assessed by using different sample types (blood and saliva) in multiple PCR and technical replicates, across different operators and different library normalization methods (magnetic beads, fluorometry and capillary gel-electrophoresis), and across two different sequencing platforms. Contamination studies were used to set thresholds for the analysis of sequencing data. We demonstrate through concordance study between MPS and Sanger results that accurate and reliable haplotypes can be obtained using our optimized Illumina protocol for whole mtDNA sequencing from reference samples.

260 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P180 - VALIDATION AND IMPLEMENTATION OF MITOCHONDRIAL DNA WITH MASSIVELY PARALLEL SEQUENCING Joseph Chang1, Matt Gabriel1, Carla Paintner1, Caron Pruiett1, Amy Liberty1

1 Thermo Fisher Scientific, Human Identification, South San Francisco, USA

Significant strides have recently been made to Next Generation Sequencing (NGS) technologies. High-fidelity chemistries allow for high multiplex assays, which drastically increase amplification sensitivity. Bioinformatic algorithm optimizations better discriminate noise versus signal, subsequently leading to accurate base calls. With all these improvements, NGS can be validated and implemented for use in forensic laboratories. Mitochondrial DNA (mtDNA) is typically utilized for the identification of ancient, disaster victim, missing person, and heavily degraded samples. Sanger Sequencing has been the validated gold standard in analyzing said mtDNA samples, however it is a long and laborious process. With the release of the Precision ID mtDNA Control Region Panel (mtDNA CR Panel) and Converge™, laboratories can process large numbers of mtDNA samples simultaneously. A validation was developed for the Precision ID System, mtDNA CR Panel, and Converge. Cur- rently published guidelines were still used, even though they are not NGS-specific. Data was generated for the following standard studies: DNA Amplification Sensitivity, Baseline/Stochas- tic, Accuracy, Precision Repeatability & Reproducibility, Mixture, and Known & Non-Probative. Additionally, 2 new studies unique to NGS, were developed: Contamination and Sequencing Sensitivity studies. A Contamination study is needed due to a combination of high mtDNA copy numbers and sensitivity of NGS systems. The results and execution of the laboratory validation demonstrated that NGS is making head- way in the forensic market. The adoption of the NGS system is no longer a daunting hurdle because the same mtDNA laboratory and interpretation guidelines can be followed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 261 P181 - VALIDATION OF A UNIVERSAL DNA EXTRACTION METHOD FOR HUMAN AND MICROBIAL DNA ANALYSIS Federica Alessandrini1, Andrea Brenciani1, Simona Fioriti1, Filomena Melchionda1, Marina Mingoia1, Gianluca Morroni1, Adriano Tagliabracci1

1 Polytechnic University of Marche, Department of Biomedical Sciences and Public Health, Ancona, Italy

The study of microbiomes has enormous potential for forensic science because microorganisms are ubiquitous and particular communities of microbes are often associated with specific processes or environments. With recent advances in microbiome science, new opportunities exist for microbiome technologies in forensic science (PMI estimation, location of clandestine graves, soil analysis and personal identification). But before new technology is accepted by the forensic science, it requires an initial validation phase. The aim of our study was to evaluate if a DNA extraction method used for human biological trace in a ISO/IEC17025 accreditated forensic lab is also suitable for microbial DNA extraction, without any modification. Several different bacterial strains (Gram-positive and Gram-negative bacteria) belonging to the most representative families recovered from human bodies and corpses were selected and submitted to the established bacterial DNA extraction protocol (GenElute Bacterial Genomic DNA Kit, Sigma-Aldrich) and to the casework DNA extraction protocol used in the forensic genetic lab of Ancona. Sensitivity, ripetibility and reproducibility of both methods were tested, both for single bacterial strain and for mixed specimens. Extracted DNA was quantified and submitted to NGS analysis on an Ion S5 NGS System. Data were analyzed using the Ion Re- porter Software metagenomics workflow. Our work has shown that it is possible to purify both microbial and human DNA using the casework extraction method developed for human DNA, thus making it possible to analyze both human and microbial DNA from a single trace, a pivotal factor in forensics where the quantities of biological material available are usually very limited.

262 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P182 - VALUE OF MASSIVE PARALLEL SEQUENCING IN POSTMORTEM GENETIC ANALYSES OF SUDDEN UNEXPECTED DEATH CASES Stefanie Scheiper1,2, Britt-Maria Beckmann3, Constanze Niess1, Christof Geisen2, Theresa E. Groß4, Peter Schneider4, Marcel A. Verhoff1, Silke Kauferstein1

1 Institute of Legal Medicine, University Hospital Frankfurt- Goethe University, Frankfurt am Main, Germany 2 German Red Cross Blood Center, Institute of Transfusion Medicine and Immunohaematology, University Hospital Frankfurt, Frankfurt am Main, Germany 3 Department of Medicine I, University Hospital Munich, Ludwig Maximilians University, Munich, Germany 4 Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany

Cases of sudden cardiac death (SCD) in young and apparently healthy individuals represent a tragic event for those left behind. Hereditary arrhythmia syndromes, which include primary electrical heart disorders as well as cardiomyopathies, are known to contribute to a significant number of these sudden death cases. Unfortunately, sudden unexpected death often reveals the first sign of a hereditary disease. We performed postmortem genetic screening in > 50 sudden death cases as well as in individuals exhibiting a family history of sudden death. The samples were sequenced by means of a defined gene panel using massively parallel sequencing (MPS). Bioinformatic analysis of the resulting data revealed several sequence variants. These were assessed according to the ACMG standards using common databases and applying in silico prediction tools. In this study, several potentially causative sequence variations could be identified in the genes analyzed. Using MPS implies challenging assessment of unknown and unclassified variants. Therefore, whenever possible, the genetic findings should be assessed in context with the medical and family history, the circumstances of death as well as autopsy findings. With careful evaluation of the genetic findings, MPS represents an important technology investigating cases of sudden unexpected death. Postmortem genetic analyses may be the only opportunity to identify an underlying genetic predisposition to SCD, which is also of high relevance in order to identify at-risk relatives and implement preventive measures in affected families.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 263 P183 - VERIFICATION OF TRANSLOCATION BETWEEN X AND Y CHROMOSOME Haijun Han1, Chunfeng Shi2, Hai Yi1, Dan Guo3, Yifan Li4, Zailiang Yu3, Chuguang Chen4

1 Nantong Public Security Bureau, Department of Forensic Medicine, Nantong, China 2 Qidong Public Security Bureau, Department of Forensic Medicine, Qidong, China 3 Suzhou Microread Genetics Co.- Ltd, Suzhou Microread Genetics Co.- Ltd, Suzhou, China 4 Beijing Microread Genetics Co.- Ltd, Beijing Microread Genetics Co.- Ltd, Beijing, China

Amelogenin is encoded by the Amelogenin gene, which has two distinguishable forms: the X-chromosome form (AMELX) and the Y-chromosome form (AMELY). Thereby, it is widely used as a sex identifier in individual identifications. Here, we reported a case where the STR profile of a female sample showed the presence of AMELY gene. Towards this abnormal phenomenon, we carried out SRY gene testing as well as the gene mapping analysis with specifically designed primers for the transposition site. Our result indicated that there was an inherited transposition between the X- and Y-chromosome in the genome of the female sample.

264 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P184 - X-INDELS EFFICACY EVALUATION IN A CRITICAL SAMPLES’ PATERNITY CASE: A SPANISH CIVIL WAR CASE FROM THE MEMORIAL OF THE CAMPOSINES (TARRAGONA, SPAIN) Cláudia Gomes1,2, Sara Palomo-Díez1,2, Carlos Baeza-Richer1,2, Ana María López-Parra1,2, Ivon Cuscó3, Caterina Raffone3, Ana Maria Cordero3, Andrea Fernández-Vilela4, Diego López-Onaindia4, Ares Vidal Aixalà5, Óscar Escala Abad5, Gemma Domenech Casadevall6, Juli Cuéllar Gisbert6, Eduardo Arroyo-Pardo1,2

1 Facultad de Medicina Universidad Complutense de Madrid, Legal Medicine- Psychiatry and Pathology, Madrid, Spain 2 Instituto de Investigación Sanitaria del Hospital Clínico San Carlos IdISSC, Grupo de Ciencias Forenses: Genética y Toxicología forenses, Madrid, Spain 3 Hospital Vall Hebron, Clinical and Molecular Genetics Area, Barcelona, Spain 4 Universitat Autònoma de Barcelona, GREAB Grup de recerca en Antropologia Biològica. Unitat d’Antropologia Biològica. Facultat de Biociències, Barcelona, Spain 5 Iltirta Arqueologia SL- Corbins, Iltirta Arqueologia, Corbins, Spain 6 Generatitat de Catalunya. Departament de Justícia, Direcció General de Memòria Democràtica, Barcelona, Spain

The Memorial of the Camposines (la Fatarella, Tarragona, Spain) consists in a place that honours all combatants of the Spanish Civil War (1936–1939), without distinction of sides. The site was included in the research carried out by the multidisciplinary team coordinated by the program of the “Direcciò General de Memòria democràtica. Departament de Justícia” (Generalitat of Cat- alonia, Spain), for the identification of the victims of the Spanish Civil War. The case investigated on the present study consisted on a presumable paternity. To accomplish the study of the presumptive father critical samples, one of the combatants found on the Memorial, two bone samples were selected - a petrous portion of left temporal bone, and a condyle of foramen magnum, both fractured and at a very bad stage of conservation. After a specific DNA extraction for critical bone samples (Gomes and Palomo-Díez et al., 2015), the next step was an autosomal and Y-chromosome analysis, trying to find a possible coincidence with any possible familiar, from the familial database. A potential match was found, indicating a possible living daughter. However, between both autosomal profiles one Mendelian incompatibility was detected, and also several allelic dropout phenomena in the presumptive father’s profile. Therefore, samples were analysed for a 32 X-chromosome Insertion-Deletion (InDel) polymorphisms battery (Pereira et al., 2012), assessing their ability to solve, or at least complementing autosomal information, in a complex paternity case. It was possible to confirm the efficacy of these 32 X-InDels polymorphisms, by solving undoubt- edly the case. Due to the 6 incompatibilities detected between both X-InDels profiles, it was now possible to discard immediately a possible paternity case.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 265 P185 - Y-STR HAPLOTYPING USING DIRECT PCR ENRICHMENTS IN MASSIVELY PARALLEL SEQUENCING James Lee1

1 National Taiwan University, Department of Forensic Medicine, Taipei, Taiwan Province of China

Library preparation for targeted sequencing with enrichment PCR in massively parallel sequencing is a time-consuming procedure. Factors include PCR amplicon purification, adapter ligations and attachment of indices. In order to minimize these procedures and reduce the risk of allelic dropout, the Illumina overhang adapter sequence to 40 Y-STR locus‐specific primers were added for PCR amplification. The primers were adapted for almost all the loci from those used in commercially available Y-STR multiplex kits. The resulting PCR amplicons were purified and indices attached for each sample. The index tagged amplicons were sequenced on MiSeq platform using MiSeq Reagent Kit v3 (Illumina) and amplified as a multiplex PCR assay. Data from all 40 regions of Y-STR loci were generated and confirmation of the Y-STR haplotypes was obtained using standard capillary electrophoresis. The results to be shown highlight the efficiency of Y-STR haplotyping using direct PCR enrichments in massively parallel sequencing.

266 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P186 - Y-STR PROFILES DETECTABLE IN FEMALE RECIPIENT’S PLASMA AFTER KIDNEY TRANSPLANTATIONS FROM MALE DONORS Eva-Maria Dauber1, Dagmar Kollmann2, Gabriela Berakovich3, Wolfgang R. Mayr4

1 Medical University of Vienna, Blood Group Serology and Transfusion Medicine, Vienna, Austria 2 Medical University of Vienna, Department of Surgery- Division of Transpantation Surgery, Vienna, Austria 3 Medical University of Vienna, Department of Surgery- Division of Transplantation Surgery, Vienna, Austria 4 Medical University of Vienna, Department of Blood Group Serology and Transfusion Medicine, Vienna, Austria

Kidney transplantation is a routine therapy of end-stage kidney failure. As gender is not a selection criterion for kidney donors, unlike-sex recipient/donor pairs are expected in about half of the transplants. This study investigated the presence of male donor-derived DNA in peripheral whole blood and plasma of female solid organ recipients. Cell-free DNA (cf-DNA) was extracted from plasma and total (predominantly nucleic) DNA from whole blood of the same blood sampling. Y-STR profiling (AmpFlSTR Yfiler kit, Life Technologies) was performed in cf-DNA samples of three female recipients transplanted with male donor kid- neys. Additionally, donor’s pre-transplant nucleic DNA was investigated as a reference for donor identification. Donor-derived Y-STR profiles were detected in the plasma of all female recipients. Peak heights continously decreased with increasing amplicon sizes and locus drop-out was observed in the upper allelic size range. In the predominantly nucleic DNA extracted from whole blood of the same blood sampling, however, no Y-STR profile was detected. These observations show, that the DNA of solid organ donors is in principle present in the whole blood of transplant recipients. It is relatively enriched in the cf-DNA extracted from plasma in a high background of recipient-specific cf-DNA. In DNA extracted from whole blood, however, donor-specific DNA was below the detection limit. By investigating cell-free recipient’s DNA, however, a low percentage of donor-derived cell-free DNA has always to be taken into consideration, which can be increased in graft rejection (Kollmann et al. 2017) or damage. Thus, solid organ transplantations should be kept in mind in the forensic field as a possible source of non-recipient DNA.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 267 POPULATION GENETICS AND FORENSIC DNA DATABASES

P187 - A COMPLICATED FULL SIBLING IDENTIFICATION CASE SOLVED BY THREE METHODS He Re1, Zhiyong Liu2, Man Chen3, Jing Zhao4, Feng Cheng5, Chen Li6, Chuguang Chen6, Yacheng Liu7, Jiangwei Yan5

1 Beijing Police College- Beijing 102202- PR China, Beijing Police College, Beijing, China 2 University of Chinese Academy of Sciences- Beijing 100049- PR China., Beijing Institute of Genomics- Chinese Academy of Sciences- Beijing- China, Beijing, China 3 University of Chinese Academy of Sciences- Beijing 100049- PR China, Beijing Institute of Genomics- Chinese Academy of Sciences- Beijing- China, Beijing, China 4 Chinese Academy of Sciences- Beijing- China, Beijing Institute of Genomics, Bijing, China 5 Shanxi Medical University- Taiyuan 030001- China, School of Forensic Medicine, Taiyuan, China 6 Beijing Microread Genetics Co.- Ltd- Beijing 100044- China, Beijing, China 7 Beijing Tongda Shoucheng Institute of Forensic Science, Beijing 100192- PR China., Beijing, China

Full sibling (FS) identification is commonly encountered in daily practice. The FS industry standard named IBS (identity by state) based on scores of shared alleles between individuals in China is ibs≥42 scores, when error rate ≤ 0.001 % and 39 autosomal STRs used. According to the standard, the vast majority of cases are accomplished successfully. Here, we report a FS identification case that the IBS scores are only 39 after typing 39 autosomal STRs loci. Therefore, ITO and IBS based on heterozygosity methods are utilized as supplement simultaneously. Fortunately, full sibling (FS) value reaches to 2.21×104 which exceeding half sibling (HS) value (1.15×103) in ITO method, and the result of IBS based on heterozygosity supports full sibling conclusion (FS/HS ratio is 2.71). The genotypes from mitochondria DNA and Y-STRs also increase persuasiveness for drawing ultimate positive identification. In practice, an exclusion conclusion shouldn’t be drawn hurriedly, although IBS scores are less than 42. The multiple kits and statistical methods applied could be available when similar condition appeared.

268 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P188 - A LOOK OF PATERNAL ANCESTRY IN A SAMPLE OF ECUADORIAN “MESTIZO” POPULATION ANALYZED THROUGH POWERPLEX Y23 German Burgos Figueroa1, Alejandra Garzón-Salazar2, Kelly Michelle Maldonado-Uquillas2, Camilo Ávila2, Michelle Adelina Toscano2, Elius Paz-Cruz3

1 Escuela de Medicina- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 Carrera de Biotecnología- Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas UDLA, Quito, Ecuador 3 Laboratorio de ADN, Fiscalía General del Estado, Quito, Ecuador

Ecuadorian population is mainly composed of Mestizos, an ethnic group whose origins date back to admixture events in post-Columbian periods, including settlements of natives, Europe- an colonization and the forced migration of African slaves. Hence, the importance to elucidate its genetic complexity and increase Ecuadorian registry, in order to help future forensic analysis and evolutionary studies. We analyzed 101 unrelated male individuals of Mestizos living scattered in the three main continental regions of the country. DNA samples were amplified using PowerPlex Y23 system and fragments were separated on an ABI Prism 3130 Genetic Analyzer. Allele sizing and profiles were obtained using GeneMapper v.3.2 and the resulting genotypes were submitted into 4 different Y-haplogroup predictors (Hapest, Nevgen, YHDR, Jim Cullen) for subsequent comparison and accurate prediction. Indeed, predictors are free tools employed before selecting more sensitive techniques like SNaPshot (system that confirms the haplotype by detecting specific mutations), saving not only time but also money. Ninety eight haplotypes observed were unique, therefore no matches were found when compared to another ones reported in YHDR Ecuadorian Database. A comparison mediated by the rest of haplogroup predictors revealed similar percentages among them, but in very specific cases we conclude that the lack of 23 markers in a predictor could lead to a false discernment. We found native lineages Q (41 %), and non-native-American lineages (59 %) represented by European lineages R1b, I (37 %); Middle East lineages G2a, T, J (8 %), and African lineages E1b1 (14 %); interestingly, African ones were found in coast provinces that weren’t assumed to have Afro settlements.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 269 P189 - A Y-CHROMOSOMAL STUDY OF SOUTHERN MANSI POPULATION GROUP IN THE URAL Horolma Pamjav1, Eszter Dudás1, Anna Vándor2

1 Hungarian Institute for Forensic Sciences- Institute of Forensic Genetics, Department of Reference Samples Analysis, Budapest, Hungary 2 Hungarian National Organization of World Congress of Finno-Ugric Peoples, Finno-Ugric Peoples, Budapest, Hungary

Based on genetic studies published, the Hungarian Y-chromosomal gene pool significantly differs from other Uralic-speaking populations. Hungarian males possess a high frequency of haplogroup R1a-Z280 and a low frequency of haplogroup N-Tat, which is common among other Uralic-speaking populations. Previously we published the Northern Mansi Y-chromosomal data and now further studied to search the links between the linguistically related Hungarian and Southern Mansi populations. Samples were collected from 36 Southern Mansi males in the Ural region. We analysed male-specific markers including Y-STRs and Y- SNPs, which would reflect past and recent paternal genetic history. In the case of the Southern Mansi males, the most frequent haplogroups were N1b-P43 (33 %), N1c-L1034 (28 %) and R1a-Z280 (19 %). On the MDS plots constructed from Fst- and Rst-genetic distances, the Southern Mansi population group showed close genetic affinities with the Khanty, Northern Mansi, Mari and Estonian populations. For phylogenetic studies, networks were constructed for the most frequent haplogroups in the Southern Mansi together with other Eurasian populations. The Southern Mansi population shared common haplotypes within haplogroups R1a-Z280 or N-L1034 with Hungarian speakers, which may suggest that the Hungarians were in contact with the Mansi people during their migration to the Carpathian Basin.

270 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P190 - ADMIXTURE AND POPULATION STRUCTURE IN MAYAS AND LADINOS FROM GUATEMALA BASED ON 15 STRS Hector Rangel-Villalobos1, José Alonso Aguilar-Velazquez2,3, Stephenson-Ojea Mishel4, Marco Antonio García-King4

1 Universidad de Guadalajara, Instituto de Investigación en Genética Molecular CUCI-UdeG, Ocotlán- Jalisco, Mexico 2 Universidad de Guadalajara, Doctorado en Genética Humana CUCS-UdeG, Guadalajara- Jalisco, Mexico 3 Universidad ed Guadalajara, Instituto de Investigación en Genética Molecular CUCI-UdeG, Ocotlan- Jalisco, Mexico 4 Fundación de Antropología Física de Guatemala FAFG, Laboratorio de Genética, Guatemala, Guatemala

Guatemala is a Central American country with a large Native-American component whose genetic structure and admixture has been poorly studied. We made a literature compilation of Guatemalan population databases for 15 autosomal STRs (AmpFlSTR™ Identifiler™ kit), which included 581 unrelated Mayan volunteers from nine indigenous groups (Ixil, Poqomchi, Achi, Qa- njobal, Poptí, Kaqchiquel, Kiché, Mam and Quechi), and 130 unrelated Ladinos (admixed). Pop- ulation structure analysis showed low differentiation among Ladino and Mayan populations of Guatemala (Fst= 0.74 %), which did not increase importantly when Ladino were excluded (Fst= 0.78 %). Admixture analysis showed that in the best-fit number of clusters (K= 3), the Asian-derived (Native-American) component is the largest contributor to the genetic pool of Guatema- lan populations (71–96 %). Although European ancestry was widely observed (2–28 %), this was lower than in other Latin American populations. Lastly, a low African ancestry was observed only when four or more clusters were analyzed (K≥ 4), suggesting a poor African contribution in the genetic pool of Guatemalan populations (<3 %).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 271 P191 - ALLELE FREQUENCIES OF 22 AUTOSOMAL STR LOCI IN POPULATION OF 1000 INDIVIDUALS FROM SOUTHEASTERN SERBIA Dijana Takić Miladinov1, Dejan Šorgić1, Perica Vasiljević2, Aleksandra Stefanović1

1 Institute of Legal Medicine, DNA laboratory, Niš, Serbia 2 Faculty of Science and Mathematics, University of Niš, Department of Biology and ecology, Niš, Serbia

The detection of polymorphism in short tandem repeats (STR) is an important method for studying variability in human populations. Allele frequencies and forensic parameters were obtained for 22 autosomal STRs (D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045) in 1000 unrelated individuals from southeastern Serbia, using FORSTAT and Arlequin softwares. A total of 284 alleles were detected with corresponding allelic frequencies ranging from 0.0005 to 0.365. No significant deviations from Hardy-Wein- berg equilibrium (HWE) were observed. Both the combined power of discrimination (PD) and the combined power of exclusion (PE) for the 22 analyzed loci were higher than 0.999999. The combined match probability for all 22 loci was 6.773688e-29. Based on heterozygosity and the polymorphism information content, as measures of informativness, D1S1656 and Penta E may be considered as the most informative markers for forensic testing. The least informative marker is TPOX. Results suggested that the 22 STR loci are suitable for personal identification in forensics and paternity testing. The calculated allele frequencies were compared with the data from neighboring populations on Balkan Peninsula.

272 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P192 - ALLELE FREQUENCY DATA FOR 15 AUTOSOMAL STRS AND ANCESTRAL PROPORTIONS USING AIMS-INDELS IN THE SHUAR ETHNIC GROUP FROM ECUADOR César Paz-y-Miño1, Odalis Astudillo González1,2, Diana Maldonado Oyervide1,2, Andrés López-Cortés1, Andy Pérez-Villa1, Isaac Armendáriz-Castillo1, Jennyfer M. García-Cárdenas1, Santiago Guerrero1, Patricia Guevara-Ramírez1, Verónica Yumiceba1, Ana Karina Zambrano1, Paola E. Leone1

1 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo- Universidad UTE, Quito, Ecuador 2 Universidad de Guayaquil, Unidad de Posgrado Investigación y Desarrollo, Guayaquil, Ecuador

Ecuador, located in South America, is known for its heterogeneous population and for being home of 21 ethnic groups, among these the Shuar, an isolated ethnic group due to geographic, linguistic and cultural factors which envelop a strong sense of identity. In the Ecuadorian amazon region, 80 thousand Shuar are settled, yet only in the Sucumbios province there are 22 thousand. In order to identify their genetic composition, 46 ancestry-informative markers (AIMs-INDELs) were used, in addition, for the first time, 40 Shuar individuals were characterized, through 15 tandem repeats (STRs) included in the Identifiler Kit, from the Kumbatza and Yu- kuteis Shuar communities in the Morona Santiago province. Results showed a non-significant Hardy-Weimberg equilibrium for 13 STRs; several markers showed linkage disequilibrium due to endogamy. From the Identifiler Kit, the multiplex PCR of the 15 autosomal STRs has demonstrated a matching probability of 0.1535, power of discrimination of 0.8465, polymorphism information content of 0.6584, probability of exclusion of 0.415 and typical paternity index of 1.78. The ancestry markers determined that the Shuar ethnic group is not influenced by admixture population events, hence, being a native American group 98.7 %, along with a genetic diversity of 0.699346+/-0.356964. Isolation of this ethnic group has enabled the conservation of their genetic material. These genetic characterization data are imperative to be considered when predicting clinical studies in the Shuar ethnic group and to elucidate about processes of evolution, migration and settlements in the history of the Ecuadorian population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 273 P193 - AN APPROACH TO MATERNAL ANCESTRY IN A SAMPLE OF ECUADORIAN “MESTIZO” POPULATION BY SEQUENCING THE CONTROL REGION OF mtDNA German Burgos Figueroa1, Filipa Simão2, Rodrigo Flores-Espinoza3, José Ignacio Yepez-Santos3, Alejandra Garzón-Salazar3, Elius Paz-Cruz4, Byron Freire-Paspuel5, Elizeu Fagundes de Carvalho2, Leonor Gusmão2

1 Escuela de Medicina- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 UERJ - State University of Rio de Janeiro, LDD - DNA Diagnostic Laboratory, Rio de Janeiro, Brazil 3 Carrera de Biotecnología- Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas UDLA, Quito, Ecuador 4 Laboratorio de ADN, Fiscalía General del Estado, Quito, Ecuador 5 Laboratorios de Investigación, Universidad de Las Américas UDLA, Quito, Ecuador

Ecuador is a country inhabited by three main ethnic groups: Mestizos, Native Americans and Afro-descendants, with varying levels of admixture between settlers from European, African, and Native American groups. Mestizos comprise about 60 % of the population and are mainly derived from the admixture between European (mostly Spanish) and Indigenous populations dispersed throughout the country. In order to improve mtDNA databases for forensic casework in Ecuador, we sequenced the mtDNA total control region (16024–576) in 54 maternally unrelated individuals from Mestizo population, by sanger method. Sequences were edited and compared to rCRS using the SeqScape software v2.7 (Applied Biosystems), and haplotypes were determined following the ISFG recommendations. Haplogroups were assigned using EMPOP database (v4 Sep2018), and their frequencies were calculated by direct counting. Haplotype diversity and mean number of pairwise differences were calculated using the Arlequin software. Haplotype diversity obtained for mestizo sample was 0.9986 (±0.0039), and a mean number of 17.59 (±7.93) pairwise differences was observed. As described in previous reports, Mestizo population revealed a high percentage of native American maternal lineages (94.4 %), composed by haplogroups inside the branches A (25.93 %), B (29.63 %), C (22.22 %) and D (16.67 %). Small percentages of Eurasian (R0 and M33) and African (L1) lineages (1.85 % each) were also found. These results show that, from the maternal side, the Mestizo population from Ecuador retains a high Native American ancestry.

274 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P194 - AN OPTIMISED METHOD FOR THE DEVELOPMENT OF A MITOCHONDRIAL POPULATION DATABASE IN NEW ZEALAND Bethany Forsythe1, Rachel Boyle2, Janet Stacey2, Sallyann Harbison2, Lisa Melia2

1 University of Auckland, School of Chemical Sciences, Auckland, New Zealand 2 Institute of Environmental Science and Research Limited, Forensic Biology Group, Auckland, New Zealand

Nuclear DNA profiling in order to form a link between an individual and a crime sample is well established in forensic laboratories. However, for evidence types containing degraded and limited nuclear DNA, such as telogen hairs and ancient or degraded remains, mitochondrial DNA analysis may be required. Due to the costs associated with traditional Sanger Sequencing of mitochondrial genomes the technique is not in place as a forensic technique in New Zealand. The introduction of massively parallel sequencing (MPS) technologies has led to the potential implementation of the sequencing of whole mtGenomes at ESR in New Zealand. In order for mitochondrial genome sequencing to be implemented as a casework technique, a relevant population database that represents New Zealand’s population and subpopulations must be in place. The New Zealand population is a diverse one comprised of an indigenous NZ Maori population of Western Polynesian origin and more recent immigrants of Polynesian origin and Cauca- sian people mainly of Western European origin. The four main ethnic groups required for a New Zealand database include Caucasians, Eastern Polynesians, Western Polynesians (including NZ Maori) and South East Asians. Establishing a local database will provide accurate frequency information regarding an individual’s haplotype. We have optimised a method to be used to build a New Zealand population database of whole mitochondrial genomes and to sequence good quality reference samples for casework comparison. This presentation will describe the progress we have made in the sequencing of population samples using the Illumina® MiSeq® MPS platform and the insights we have gained so far.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 275 P195 - AN UPDATE OF STR MUTATION RATES OF PATERNITY TESTS STUDIED FOR 14 YEARS (2005–2018) AT IDENTIGEN LAB, UNIVERSIDAD DE ANTIOQUIA, COLOMBIA German Burgos Figueroa1, Yeny Posada2, Camilo Ávila3, Alison Florez Misas4, Adriana Ibarra2

1 Escuela de Medicina- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 IdentiGEN-Genetic Identification Laboratory and Research Group of Genetic Identification- Institute of Biology- School of Natural and Exact Sciences FCEN, University of Antioquia, Medellín, Colombia 3 Carrera de Biotecnología- Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas UDLA, Quito, Ecuador 4 Facultad de Ciencias de la Salud- Bacteriología y Laboratorio Clínico, Institución Universitaria Colegio Mayor de Antioquia, Medellín, Colombia

DNA testing is the gold standard for biological filiation and human identification, based mainly on the large number of polymorphisms of STRs, caused mainly by relatively frequent mutations, most commonly produced by polymerase slippage. Changes in the size of alleles in the STR sequences are often associated with these mutations, causing alterations in the statistical calculations of tests, thereby lowering the PI. To avoid such alterations and obtain a paternity probability (W) above 99.99 %, it is necessary to increase the number of STRs analyzed and to know the appearance frequency of these mutations. In the present study, 18 STR were analyzed in a total of 11077 cases, according to the paternity reports issued by the IdentiGEN Lab, focused mainly on the population of Antioquia, during a period of 14 years (2005–2018). DNA extraction was carried out using Chelex® and FTA® methods. Molecular marker amplification was achieved using the PowerPlex® 16 (Promega), Identifiler® (Applied Biosystems), FFFL® (Promega), and Tri- plex® (In house) kits. Detected mutations were confirmed by combined typing of the different kits described, depending on the specific requirements of the case. The amplicons were separated by capillary electrophoresis in an ABI 3130 genetic analyzer (App. Biosys. ®). The allelic sizes of the STRs were assigned with GeneMapper® v3.2 software. Mutation rates were calculated by single counting. In 11077 cases, a total of 252 mutations were found; 248 were one-step mutations, 3 were two-step and only one three-step mutation at D18S51 locus was observed.

276 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P196 - ANALYSIS OF MUTATION RATE OF 13 RAPIDLY MUTATING Y-CHROMOSOME SHORT TANDEM REPEATS IN TANZANIAN FATHER-SON PAIRED SAMPLES Rashed Alghafri1, Bugoye Fidelis2, Reem Almezaina1, Sarah Khoory1, Leticia Waitara3, Elias Mulima2, Gerald Misinzo2

1 Dubai Police G.H.Q, Forensic Sciences and Criminology, Dubai, United Arab Emirates 2 Government Chemist Laboratory Authority, Department of Forensic Science and DNA Services, Dar es Salaam, Tanzania- United Republic of 3 Government Chemist Laboratory Authority, Department of Forensic Science and DNA Services, Dubai, United Arab Emirates

The interest of the forensic community has been raised lately toward Y-chromosomal short tandem repeats (Y-STRs), termed Rapidly Mutating Y-STRs (RM-YSTRs), which have been proven to differentiate between close males belonging to the same paternal lineage to some extent [1]. This is due to their high mutation rates relatively. In this study, the mutation rate for 13 RM- YSTR was estimated in 100 pairs of male relatives in the population of Tanzanian in order to evaluate the capacity of differentiating between male individuals within a single lineage. Buccal swabs were collected from consented individuals from Tanzanian population. Each father-son couple was previously confirmed by autosomal STRs testing AmpFℓSTR® Identifiler ™ kit (Ther- mofisher) with paternity probability ≥99.99 % and also was previously analyzed by AmpFℓSTR® Yfiler™ (Thermofisher). Samples were amplified using RM-Yplex amplification assay [2]. PCR products were analyzed using ABI Prism 3130XL Genetic Analyzer and analysed using Gene Mapper ID v.3.2 software. Data analysis will be carried out using the Microsoft Excel Software and the confidence interval (CI) will be estimated from the binomial standard deviation. Compared to the 17 loci available in Yfiler™ kit, it is expected to see higher level of discrimination within the same set of samples. The resulted mutation rates will be compared with previous studies of the same markers in different populations. Such study is very critical ahead of using and inter- pretating such rapidly mutating loci in forensic investigation or paternity cases involving males in order to understand the number of expected mutations between generations for Tanzanian population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 277 P197 - ANALYSIS OF PATERNAL LINEAGES LEGACY IN SALTA PROVINCE, NORTHWEST ARGENTINA Joana Francesca Ferragut1, Maria Virginia Albeza2, Noemí Acreche2, José Aurelio Castro1, Cori Ramon1, Antonia Picornell1

1 University of Balearic Islands, Biology, Palma, Spain 2 Universidad Nacional de Salta, Cátedra de Antropología, Salta, Argentina

Salta province is located in the northwest of Argentina (NWA). Numerous native people lived there before the Spanish conquest. There is no historical data regarding the population structure of region, in pre and post-contact period with Europeans. Due to this fact, the exact origin and/ or degree of admixture of Salta current inhabitants are unknown. Nowadays, they can be considered ‘mestizo’ populations, result of intermarriage between natives of the Amerindian tribes that inhabited the area before colonization and, immigrants of diverse origins and African slaves introduced in the colonial period. In the present work, we analyzed 174 non-related male individuals belonging to urban and rural areas from Salta region, aiming to investigate their paternal lineages. Seventeen Y-STRs were typed using AmpFlSTR® Yfiler® PCR Amplification Kit. One hundred and sixty-six different haplotypes were found. Forensic parameters showed high values, Haplotype Diversity was 0.9992 and the Discrimination Power was 95.4 %. Three haplogroups encompassed the 77 % of the diversity: Q (33 %), (33 %) and E (11 %). Significant differences were found between urban and rural samples, in the city the diversity was higher (99.96 % vs 97.64 %), with 8 different haplogroups and being the modal the European R1b, whereas within rural individuals the Amerindian haplogroup Q reached a frequency of 65 %, more than twice the R1b frequency. Therefore, these results are in accordance with the Amerindian legacy of these populations together with Euro- pean and African contributions.

278 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P198 - ANALYSIS OF THE COMPLETE mtDNA IN BRAZILIAN SAMPLES: HAPLOTYPES DIFFERENTIATION AND NEW VARIATIONS IDENTIFICATION IN NATIVE AMERICAN HAPLOGROUPS Danilo Braganholi1, Jorge Marcelo Freitas2, Nathália Andrekenas1, Bianca Belon Januario1, Isabela Brunelli Ambrosio1, Fernanda Silva Polverari1, Regina Cicarelli1

1 Laboratório de Investigação de Paternidade- UNESP-FCFAr, Ciências Biológicas, Araraquara, Brazil 2 Polícia Federal- Instituto Nacional de Criminalística, Brasília, Brazil

The Brazilian territory that until about the year 1500 was occupied only by Native Americans, was colonized by European navigators that brought African slaves generating one of the most miscegenated populations of the world [1]. Initial data about the population of the Center-West Brazil region for mtDNA CR were published in 2019 [2] and, to date, there is no published data of complete mtDNA for any Brazilian population. In this context, 74 samples were selected from individuals of the Center-West Brazil region who shared haplotypes by the mtDNA CR. The purpose is to analyze the complete mtDNA by Massively Parallel Sequencing and to evaluate if it is possible to differentiate these haplotypes. The libraries were performed for all samples and to date 22 samples were sequenced by the Illumina system. Analyzing only the CR, 8 haplotypes were identified in these 22 samples. The complete mtDNA analysis allowed the identification of 16 haplotypes, 6 of which were shared by two samples each, that is, the diversity of haplotypes in this sample increased. In addition, were identified some genetic variants not reported in the EMPOP: 16182del; 16178del, 16192.1T, 14881A; 15382A, 15716A. Each one of these variations was identified separately in 6 different mtDNAs, 4 of which were classified in haplogroups A2 or B2, which have Native American ancestral origin; the other 2 mtDNAs were classified into haplogroups with African ancestral origin (L2a1f and L0a2a2a). The haplogroup L2a1f has been reported to occur in African Americans [3]. It is possible that the identified variations are related to Native American haplogroups or that occur in the Americas, which should be confirmed by the analysis of more samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 279 P199 - ANALYSIS OF TWO X-STRS MARKERS PANELS IN SÃO PAULO STATE POPULATION (BRAZIL) Bianca Belon Januario1, Fernanda Silva Polverari1, Flávia Alves de Jesus Silva1, Danilo Faustino Braganholi1, Isabela Brunelli Ambrosio1, Regina Maria Barretto Cicarelli1

1 Faculdade de Ciências Farmacêuticas, Unesp São Paulo State University, Biological Sciences, Araraquara, Brazil

The analysis of DNA polymorphisms is an indispensable tool in forensic genetics. Analyses are based on the genetic profile of an individual, by combining several markers that are inherited from their parents. X-chromosomal markers can be useful in some forensic cases, where the analysis of the autosomal markers is not conclusive. In this context, sexual chromosome X analysis has improve significant importance in the forensic and population area due to its pattern of transmission between the parents. In this study, we analyze a population sample of 146 unrelated individuals from São Paulo state for two X-STRs panels: Decaplex (Gusmão, 2009) and the commercial Investigator Argus X-12QS kit (Qiagen). The cumulative discrimination power (PD) for study population with commercial kit Argus was 1 for females and 0,99999999989899 for males, and with Decaplex panel was 0,99999999995646 for females and 0,999999465754943 for males. The high PD value for Argus panel in relation to Decaplex is due to the higher number of Argus analyzed markers, which contains 12 markers, whereas the Decaplex has only 10 markers. In addition, the markers present in the Argus kit have a higher allelic variability than Decaplex markers. It may help in the future in the resolution of forensic cases and human identification, as well as to increase genetic knowledge for the São Paulo State population.

280 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P200 - ANALYTICAL IMPROVEMENTS IN BIOGEOGRAPHIC ANCESTRY INFERENCE Sharon Wootton1, Sharada Vijaychander1, Jie Deng1, Ryo Hasegawa1, Angela Lackey1, Matt Gabriel1, Robert Lagacé1, Jessica Lim1

1 Thermo Fisher Scientific, Human Identification Division HID - MPS, South San Francisco, USA

Ancestry-informative markers (AIMs) can be useful alongside phenotype informative markers to limit a suspect pool or provide investigative leads when STR profiles are either incomplete or fail to provide a database match. On next-generation sequencing (NGS) platforms, these markers can be multiplexed by the hundreds and can present a comprehensive depiction of the unknown individual’s ancestry and lineage using a consolidation of both autosomal and haploid markers. Panels including the Precision ID Ancestry Panel as well as consortia grown panels such as MAPlex and VISAGE tools have led to recent software efforts to allow for customizable marker sets and allele frequencies, and to improve biogeographic prediction accuracy and error estimation. We propose a bootstrapped maximum likelihood approach to ancestry admixture prediction and assess its ability to assign biogeographical ancestry to samples from the 1000 Ge- nomes Project and from samples typed for the ALFRED database (3). Admixed profiles were then created by simulating inheritance from randomly selected profiles, and predictions were run on the simulated offspring. Using a higher confidence interval, we demonstrate correlation between reported error and uncertainty in the prediction due to lack of differentiation. We conclude that overall, predictions are generally consistent with self-reported ancestry, and for populations with predictions of higher uncertainty, we propose inclusion of region-specific markers that can further discriminate these populations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 281 P201 - ANCESTRAL ANALYSIS OF A NATIVE AMERICAN ECUADORIAN FAMILY WITH CONGENITAL INSENSITIVITY TO PAIN WITH ANHIDROSIS Andrés López-Cortés1, Ana Karina Zambrano1, Patricia Guevara-Ramírez1, Byron Albuja Echeverría2, Santiago Guerrero1, Eliana Cabascango3, Andy Pérez-Villa1, Isaac Armendáriz-Castillo1, Jennyfer M. García-Cárdenas1, Verónica Yumiceba1, Gabriela Pérez-M4, Paola E. Leone1, César Paz-y-Miño1

1 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo- Universidad UTE, Quito, Ecuador 2 Hospital San Luis de Otavalo, Ministerio de Salud Pública, Otavalo, Ecuador 3 Sistemas Médicos SIME, Universidad San Francisco de Quito, Quito, Ecuador 4 Centro de Salud, Ministerio de Salud, Ibarra, Ecuador

Congenital insensitivity to pain with anhidrosis (CIPA) is an extremely rare autosomal recessive disorder characterized by self-mutilating behavior, unexplained fever, inability to sweat and in- tellectual disability. CIPA pathogenesis is associated with genetic loss-of-function mutations of the NTRK1 gene, which is autophosphorylated activating intracellular signaling transduction such as cell survival, growth and differentiation. Regarding to epidemiology, CIPA occurs with an incidence of 1 in 125 million newborns, and only some hundreds of cases have been reported worldwide. Most of cases have been reported in Japan, China, Korea, Sweden, Turkey, Kuwait, Italy, Spain, United States, Poland, Pakistan, Saudi Arabia, Netherlands, Malaysia, Israel, India, Germany, France, Ecuador and United Arab Emirates. In the present study, we estimate the ancestral proportions of a family with consanguinity background affected with CIPA, who car- ries the missense pathogenic mutation rs80356677 (Asp674Tyr) in the kinase domain of NTRK1. The ancestral proportion was calculated through 45 ancestry informative markers (AIMs) and the comparison was done through the Human Genome Diversity Project panel. The resulting allele frequencies in CIPA family indicate a prevalence of the Native American ancestral component with 87.9 %, and minor proportion for the European and African components with 8.9 % and 3 %, respectively. In conclusion, the genetic variations expressing CIPA in a Native American Ecuadorian family could have been caused by the insertion of certain genetic characteristics, which have been passed down from common ancestors as consequence of migration towards South America.

282 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P202 - ANCESTRAL PROPORTIONS BASED ON 22 AUTOSOMAL STR OF AN ADMIXED POPULATION (MESTIZOS) FROM PENÍNSULA DE YUCATÁN, MEXICO Lizbeth Gonzalez-Herrera1, Javier Enrique Sosa-Escalante2, Paola Lopez-Gonzalez2, Maria Jose Lopez-Gonzalez2, Rosalba Yaqueline Gamboa-Magaña3, Refugio Guadalupe Herrera- Diaz3, Karla Adriana Piña-Dzul3, Stefany Fabiola Leon-Acosta3, Rolando Ismael Flores-Baas3, José Alonso Aguilar-Velazquez4, Rodrigo Rubi-Castellanos1, Hector Rangel-Villalobos4

1 Universidad Autonoma de Yucatan, Laboratorio de Genetica. Centro de Investigaciones Regionales, Merida, Mexico 2 DIMYGEN Laboratorio SCP, Laboratorio Privado, Merida, Mexico 3 Instituto de Ciencias Forenses del Estado de Yucatan, Direccion de Quimica Y Genetica Forense, Mérida, Mexico 4 Universidad de Guadalajara, Instituto de Investigación en Genética Molecular, Ocotlan, Mexico

Latin American populations trace their ancestry mainly to the Post Columbian admixture among Native Americans, Europeans, and Africans during roughly 500 years. Although historic and anthropological records suggest that the Peninsula of Yucatan population (Southeast, Mexico) has as a large Native American origin of Mayan ethnicity, they have been poorly studied with autosomal STRs. Thus, the structure and ancestral proportions of the Mestizos (admixed population) from the Peninsula of Yucatan were assessed by genotyping 687 subjects for 22 autosomal STRs (Powerplex Phusion system). STR databases from other Mexican Mestizo populations were included for the admixture analysis, in addition to European, Native American, and African genetic pools as ancestral references. Interpopulation analysis among Mexican-Mestizos showed different allele distribution for some STRs (p<0.03): TH01, D18S51, D5S818, D7S820, and TPOX. The best fit of population clusters obtained with the Structure software (K = 5), suggests that the Mexican population of the Peninsula de Yucatán is mainly composed by Native American (64.9 %), Euro- pean (33.7 %) and African (1.3 %) ancestries.The Native American ancestry in the Peninsula of Yu- catan was the highest among the included Mexican Mestizo populations (range: 51.5 - 56.4 %) and Hispanic Americans (32.9 %). Conversely, the European ancestry in the Peninsula of Yucatan was the lowest among Mexican Mestizos (33.5 %), similar to the estimated in Mexico City (35 %), but lower than the observed in Monterrey city (40.4 %) and the West region (48.5 %). This particular landscape of ancestral proportions in Mexico justify the creation of local STR databases for human identification purposes in this country.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 283 P203 - ANCESTRY EVALUATION IN A POPULATION SAMPLE OF THE TUNJA CITY, DEPARTMENT OF BOYACÁ – COLOMBIA Izquel Sanchez1, Libardo Mendoza1, Rincon Alma Luciel2, Diana Aguirre1, Juan José Builes1,3

1 Genes SAS, Paternity, Medellin, Colombia 2 Laboratory of Genetics and Specialized Tests SAS, Paternity, Tunja, Colombia 3 University of Antioquia, Institute of Biology, Medellin, Colombia

Ancestry Informative Markers InDels (AIM-InDels) show high allele frequency variation between ancestral populations and are useful to estimate individual and population ancestry. This work determined the African, European and Native American admixture proportions in a population sample of 126 individuals from Tunja, Department of Boyacá, Colombia. The ancestral components were calculated according to the methodology described in [1]. The software Arlequin v3.5.2.2 was used to estimate allele frequency distributions, and to perform Hardy–Weinberg equilibrium analyses. The software STRUCTURE v2.3.4 was used to estimate the African, European and Native American admixture proportions in the population sample of Tunja. As parental populations, the genetic profiles of 319 individuals was used (Africans: 105, Europeans: 154, and Native Americans: 60). The ancestry analysis for this population revealed a main European contribution (55 %) with an important contribution of Native American ancestry (40,8 %) and only 4,2 % contribution of African descent. The results obtained from this type of studies allow us to have a real approximation of the state of the different Colombian populations after 50 years of war and high levels of displacement of the civilian population. 1. Pereira R, Phillips C, Pinto N, Santos C, Santos SEBd, Amorim A, et al. Straightforward inference of ancestry and admixture proportions through Ancestry-Informative Insertion Deletion multiplexing. PLoS ONE. 2012; 7: e29684. doi: 10.1371/journal.pone.0029684 PMID: 22272242

284 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P204 - ASSESSMENT OF AUTOSOMAL, Y-STRs AND MITOCHONDRIAL DNA PROFILES IN THE ZAMBIAN POPULATION (R.I.P) Kapema Bupe Kapema1, Innocent Makasa1, Nisha Thakur1

1 Ministry of Home Affairs, National forensic science authority, Lusaka, Zambia

Zambia is one of the few countries without frequency population databases on Autosomal, Y-STR and mitochondrial DNA impacting negatively on the use of DNA evidence. Statistical data is calculated using foreign databases not representative of local populations. The project is aimed at creating Zambian population databases of Autosomal, Y-STR and mitochondrial DNA comparable with other databases within the region. This will be a cross-sectional study comprising of 1500 unrelated individuals who will be sampled randomly from the six major tribes spread across the 10 provinces of Zambia. 150 individuals will be recruited from each of the ten provinces; Lusaka, Copperbelt, Luapula, Northern, North-western, Muchinga, Eastern, Central, Southern and Western. Samples will be collected by buccal swabs using Easicollect plus® kits and stored at room temperature. So far, 150 samples have been collected from the Lamba speaking people across the Copperbelt province, informed consent was obtained from the participants and brief questionnaires on their general information were completed. 15 out of the collected 150 samples were subjected to Y-STR analysis by direct PCR using PowerPlex® Y23 kit and detection of the PCR product was performed using genetic analyser ABI PRISM™ 3200 (Applied Biosystems). The predominant markers observed were DYS391 and DYS389 I at loci 13 and 10 respectively comparable to the Bakongo of Angola. The markers DYS481, DYS437, DYS385 and DYS456 had the majority haplotypes, however, the results are only qualitative due to the small sample size.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 285 P205 - AUSTRIAN STR POPULATION DATA USING THE POWERSEQ GY46 SYSTEM AND MASSIVELY PARALLEL SEQUENCING Petra Müller1, Burkhard Berger1, Martin Bodner1, Walther Parson1,2 The DNASEQEX Consortium

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

With massively parallel sequencing (MPS) technologies it is possible to characterize the sequence variation within the repeat and flanking regions of short tandem repeats (STRs) that have traditionally been analysed using fragment sizing via capillary electrophoresis (CE). To account for MPS-based allele frequencies valid for Austrians we performed a population study including 249 unrelated male donors born in Austria. Concordance to CE was assessed for all samples using the AmpFlSTR NGM Select PCR Kit on an Applied Biosystems Prism 3500 XL Genetic Analyzer using the Gene Mapper ID-X software (all Thermo Fisher Scientific, Waltham, USA). MPS libraries were manually prepared using the PowerSeq GY46 system (Promega, Madison, USA) according to the manufacturer´s recommendations. Sequencing was performed on the MiSeq FGx benchtop sequencer (Illumina, San Diego, USA) and analysed with the STRait Razor v2 software (King et al., 2017). Among the 22 autosomal STR loci considered in this study particularly the markers D12S391 and D21S11 were found to be highly variable after MPS analysis and resulted in a 3.4- fold and 2.6-fold increase with respect to the number of observed alleles compared to CE. This Austrian dataset is expected to provide sequence based allele frequencies to support the implementation of MPS into forensic routine applications. This study was part of the EU-funded project DNASEQEX (DNA-STR Massive Sequencing & Interna- tional Information Exchange). King JL, Wendt FR, Sun J, Budowle B, Forensic Sci Int Genet 29 (2017) 21–28.

286 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P206 - AUTOSOMAL STR LENGTH, REPEAT AND FLANKING REGION SEQUENCE VARIATION IN THE NORWEGIAN POPULATION USING THE MISEQ FGX FORENSIC GENOMICS SYSTEM Kirstin Janssen1, Sandra Buadu1, Nina Mjølsnes Salvo1, Marthe Aune1, Marita Olsen1, Gunn‑Hege Olsen1, Thomas Berg1

1 UiT The Arctic University of Norway, Centre of Forensic Genetics, Institute of Medical Biology, Faculty of Health Sciences, Tromsoe, Norway

Relevant population databases are necessary to calculate the statistical weight of DNA evidence. The recent increase of markers included in commercial STR-kits requires that allele frequencies of new markers are obtained for each population. Furthermore, a potential move to massively parallel sequencing in casework requires that sequence-based genotyping results are validated against existing CE-based STR-typing results and that detailed knowledge about sequence variation is obtained. In this study, we used the MiSeq FGx Forensic Genomics System to examine both the repeat length as well as repeat and flanking region sequence variation in more than 500 individuals of the Norwegian population. Sequence variation in flanking regions was analyzed with the recently released Universal Analysis Software v1.3 (Verogen). Length-based genotypes were compared to genotypes obtained with the CE-based AmpFL- STR™ NGM SElect™ PCR Amplification Kit (Thermo Fisher Scientific). Genotypes were 99.99 % concordant in the 15 overlapping markers. For the majority of markers, the number of alleles increased considerably when including sequence variation, and novel sequence-based alleles were discovered in comparison to online databases and previous publications. The Norwegian population data will be submitted to relevant allele frequency databases.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 287 P207 - CAN THE ANALYSIS OF THE Y CHROMOSOME HAPLOTYPES BE HELPFUL IN EXPLAINING THE MYSTERY OF THE FIRST SLAV MIGRATION? Jakub Czarny1, Monika Antczak1, Bartosz Hornik1, Joanna Idkowiak1, Łukasz Kaczorowski1, Aleksandra Górka1, Jolanta Powierska-Czarny1, Magdalena Chrostowska1, Zuzanna Czarny1

1 Institut of Forensic Genetics, Department of Forensic Genetics, Bydgoszcz, Poland

Analysis of Y-STR loci polymorphism in 600 men from 8 historical regions of Poland was conducted and Y chromosome haplotypes were determined. The R1a haplotype predominated (>50 %) in all regions. Other haplotypes were found at a much lower frequency. The clearly major character of one haplogroup may be related to the arrival of Slavs in Central and Eastern Europe regions, previously abandoned by Germanic tribes. The shape of modern Europe is the result of many migrations, but migrations in the IV - IX centuries were most important. This period includes numerous migrations of Germanic tribes, Franks and Anglo-Saxons, invasion of Huns, the arrival of Slavic tribes and Viking raids. These processes ranged from invasions to the transfer of elite, but situations when newcomers were sent to areas abandoned by earlier residents were rare at that time. It is assumed that the arrival of Slavs in Europe occurred in the VI century and is associated with the appearance of ceramic cultures in current regions of e.g. Czech, Poland, Belarus and Ukraine. The ceramics of Slavic tribes were produced without the use of a potter’s wheel and, similar to the metallurgical products, are distinguishable from the achievements of Germanic tribes which formerly inhabited these lands. The Slav settlements and their homes were also more primitive. Palynological analyses also indicate a strong breakdown of crops during the arrival of the Slavs, which suggests that in many aspects they started ab ovo. The results of the Y chromosome haplotype analysis of contemporary inhabitants of Poland are another confirmation of the hypothesis that earlier collapse of Germanic settlements in the areas occupied by the arriving Slavs was associated with significant depopulation of these areas.

288 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P208 - CHARACTERIZATION FROM NORTHERN SOUTH AMERICA POPULATIONS REVEALS THE NEED FOR THE SUBSTRUCTURE ESTIMATORS USE IN FORENSIC STATISTICS William Usaquén Martínez1, Andrea Casas-Vargas1, Fernanda Mogollón Olivares1, Sara Zea Montoya1, Julie Moncada Madero1, Dayana Suárez Medellín1, Leonor Gusmão2

1 Universidad Nacional de Colombia, Institute of Genetics, Bogotá, Colombia 2 Universidade do Estado de Rio de Janeiro, Laboratório de Diagnóstico por DNA, Rio de Janeiro, Brazil

Colombia, in the north of the South American continent is a country of great interest in genetic studies owing to its high ethnic and cultural diversity. This diversity is represented by its 87 native-american, and african-american populations, ROM or gypsy people, 64 amerindian languages and a great diversity of dialects grouped into 13 linguistic families. In the present study an analysis of the genetic structure and ancestral composition was carried out through a panel of 46 INDELS-AIMS ancestry information markers, taking into account the demographic and genealogical variables of 489 unrelated individuals sampled in ten years, representing 15 native-american, 2 afrodescendant and 5 mixed-ancestry communities, obtaining as a result the model of three type-populations, which was also revealed with STRs markers. Due to this, we suggest that substructure values should be considered when performing forensic statistics, especially for Colombian population due to the situation of violence in the country, where crimes against humanity have been committed against indigenous communities and afro-descendant groups. In this way we would achieve a substantial improvement in the probability values we report in this type of case.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 289 P209 - CHUETA POPULATION: DIVERSITY AND FORENSIC PARAMETERS OF AN SMALL, ISOLATED AND ENDOGAMIC POPULATION Joana Francesca Ferragut1, José Aurelio Castro1, Cori Ramon1, Antonia Picornell1

1 University of Balearic Islands, Biology, Palma, Spain

Chuetas are the descendants of the condemned crypto-Jews in the last Inquisition processes in Majorca (Balearic Islands, Spain) in the 17th century. Despite their conversion to Catholi- cism, they were discriminated against and secluded by their immediate neighbours. Since they have always lived in a small island, they have been also recognised by everyone in the island. Therefore, Chuetas are a reduced group, isolated not only by their social discrimination but also geographically. Consequently, they have had strong endogamic behaviour until the middle of the twentieth century. These characteristics made the Chuetas an interesting group of study since the genetic diversity was expected to be considerably low. Contrary to this, diversity found in different markers (autosomal STRs and Indels, mitochondrial DNA, X-chromosome STRs, Alus and Indels and Y-chromosome STRs and SNPs) show that the diversity in this population is as high as in other Jewish or islander populations studied to date. There is only one forensic parameter that is significantly low: discrimination capacity in Y-chromosome STR haplotypes, which is 53 %. These results suggest that probably the ancestral Majorcan Jewish population, which originated the Chueta group, was genetically very diverse. Moreover, mating strategies can have allowed not losing their diversity during the isolation period.

290 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P210 - CODIS-LIKE CLOUD EXPERT SYSTEM FOR HANDLING AND EXCHANGE OF THE NON-HUMAN DNA STR ANALYSIS RESULTS Daniel Vanek1,2,3, Pavla Rihova4, Edvard Ehler1, Simona Dalihodova1, Zdenek Strnad5

1 Forensic DNA Service, DNA laboratory, Prague, Czech Republic 2 Charles University in Prague, 2nd Faculty of Medicine, Prague, Czech Republic 3 Institute of Legal Medicine- Bulovka Hospital, DNA laboratory, Prague, Czech Republic 4 Czech Environmental Inspectorate, Cites, Prague, Czech Republic 5 Netsystems- a.s., Headquarters, Liberec, Czech Republic

Poaching animals for use in traditional medicines and illegal trade have a huge a negative impact on critically endangered animals protected by the CITES Convention. DNA barcoding techniques providing information about the specimen identification within the tested sample are established methods offering qualitative information about the matching organism despite some pending problems. The investigation of wildlife crime based just on specimen identification would not be in many case sufficient as individual identification or paternity is often the key information enabling to solve the case. Human STR based DNA identification for forensic purposes is well established method offering the comparison of DNA profiles nation- and world-wide and to perform familiar searches using CODIS software. Forensic animal DNA typing on the other hand is still far away from the standardization level of human DNA identification. The recommendations of ISFG serve as a good lead for the development of STR typing systems but unfortunately there are practically no scientific papers describing fully validated STR typing multiplexes or CODIS-like databases for CITES organisms involved in wildlife crime. Also there is a lack of knowledge base like NIST STRBase or CODIS-like database. The aim of this talk is to present the novel Cloud Expert System for handling and exchange of the non-Human DNA STR analysis results. The Cloud Expert System (CES) combines the cross-ref- erenced registries of STR markers for different species, DNA database, repository of scientific papers and a dashboard for unpublished data, protocols, negative results and announcements related to animal DNA typing.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 291 P211 - COMPARISON OF GLOBALFILER™ PCR AMPLIFICATION KIT AND PRECISION ID GLOBALFIER™ NGS STR PANEL – IMPLICATIONS ON FORENSIC CASEWORK Milica Keckarevic Markovic1, Vanja Tanasic1, Milica Mihajlovic1, Verica Radojicic1, Fedja Puac1, Miljana Kecmanovic1, Dusan Keckarevic1

1 University of Belgrade, Faculty of Biology, Department for Biochemistry and Molecular Biology, Belgrade, Serbia

Massively parallel sequencing (MPS) is a breakthrough methodology that has been moving boundaries in human genetic research, especially in diagnostics of rare diseases, cancer research, developmental disorders, and, also, changing paradigms in basic science. The principle of MPS allows parallel sequencing of many different relatively short DNA fragments, analyzing in the same time the length and structure of STRs and SNVs in the flanking sequences. STR loci analyzed by MPS show higher diversity than the same loci analyzed by standard CE methodology, there are almost no constraints in number of loci analyzed in the same time, and there are fewer constraints in minimal amplicon lengths. As a consequence, MPS could, in particular, be helpful in analyses of degraded samples, as well as mixture analyses, in which it could be used in differentiation of minor contributor from stutter artifact, as well as in morereliable estimation of number of contributors. In order to start using MPS with all its advantages, in routine forensic DNA analysis, a reliable population based allelic frequency DNA database should be created. Here we present the results of study done for 22 forensic loci shared by GlobalFiler TM PCR Amplification Kit and Precision ID GlobalFiler TM NGS STR panel, which showed 100 % concordance. Calculated allelic diversities were shown to be higher for Precision ID GlobalFiler TM NGS STR panel, as it was expected. Also, MPS was more reliable in distinguishing drop-ins from general artifacts than CE analysis, allowing better estimation of the probability of drop-ins. Obtained DNA profiles are meant to be used for creating MPS STR allelic frequency data for Serbian population, as condition sine qua non for full capacity introduction of MPS in forensic casework.

292 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P212 - COMPARISON OF MINIDOGFILER AND “ASCH” STR MULTIPLEX SYSTEMS FOR PRELIMINARY ESTIMATION OF VARIABILITY WITHIN WOLF´S LIKE DOG BREEDS Radka Štikarová1, Jakub Vašek1, Eniko Kubinyi2, Pavel Vejl1, Daniela Čílová1

1 Czech University of Life Sciences Prague, Department of Genetics and Breeding, Prague, Czech Republic 2 Eötvös Loránd University, Department of Ethology, Budapest, Hungary

This study was undertaken to determine preliminary statistic results in population parameters in comparison of two STR identification panels. Wolf´s like dog breeds and wolves samples were selected for their close genetic relationship. The total number of used samples were the Eurasian wolf (n=21), Czechoslovakian wolfdog (n=55), Saarloos wolfdog (n=22) and German Shepherd (n=26). Identification panels Mini-Dog- Filer and „ASCH“ panel were selected for analyses purposes. They were used separately and together for a statistic evaluation for all sample groups. Totally 228 alleles were detected using 20 SSR markers (Mini-DogFiler 11 and „ASCH“ 9 loci). Numbers ranged from 3 to 23 per locus, with median 11 alleles per locus for Mini-DogFiler and 10 for „ASCH“. Locus REN214L11 had the lowest number of identified alleles. In contrast, locus FH3210 and FH2361 showed large polymorphism, with 23 and 22 alleles identified. Thess markers were part of the „ASCH“. VGL1165 was with 20 alleles the most variable locus found in the Mini-DogFiler panel. A group of wolves showed high variability occurrences of identified alleles, unlike Saarloos wolfdog who showed the lowest variability for all analyses. Modelling based on Bayesian techniques using STRUCTURE programme were used for group number verification, which confirmed presence of 4 groups in all cases. This study could allow the empiric estimation of actual frequencies on individual alleles, which can be used to calculate the probability of random matching of two genetic profiles and an analogy to the use of such information in forensic genetics. On the base of these analyses allelic ladders and standards were developed, which are used to evaluate much more genotype.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 293 P213 - COMPARISON OF Y-STR HAPLOTYPES IN IRANIAN, AFGHAN, AND MONGOLIAN POPULATIONS Atefeh Joudaki1, Sirous Zeinali2

1 Nagoya University School of Medicine, Advanced Medical Science, Nagoya, Japan 2 Kawsar, Human Genetics Research center, Tehran, Islamic Republic of Iran

By using KBC Y-Filing Kit (Kawsar Biotech co, Iran), approximately 450 Iranian males belonging to 10 ethnic groups were profiled by 17 Y-STRs and compared with 201 Iranian samples which had been published before by other researchers. To have a better view of the Iranian population, we made a comparison between 56 male samples from Iraq, which had it been profiled in our lab, in combination with 201 Afghan male profiles and 259 Mongolian male profile which had been profiled by other researchers before. We conducted 15 Y-STRs Median-joining networks combining the different ethnic groups to disclose a broader range of diversity values in the network structures. C3 haplogroup of the Af- ghan Population including Uzbak and Hazara Ethnic groups were is significant at the presence of the star-like structure in combination with Ordos-Western Inner Mongolian population. Masal population in the north of Iran had a significant structure. Ethnic groups of east and west of Iran can be divided into two different groups.

294 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P214 - COMPLETE MITOCHONDRIAL GENOME VARIATION IN SWISS POPULATIONS USING THE ION S5 Mario Gysi1, Cordula Haas1, Natasha Arora1, Christina Strobl2, Adelgunde Kratzer1, Walther Parson2,3

1 Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland 2 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 3 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

Whole mitochondrial genome (mitogenome) sequencing has shown to significantly increase the discrimination power for forensic samples or kinship cases compared to conventional control region sequencing. However, haplotype frequency estimates rely on control region population data as the number of forensic reference full mitogenomes is still limited in the mtDNA database EMPOP. To contribute to an increasing database of full mitogenomes we used the Pre- cision ID mtDNA Whole Genome PanelTM and the Ion S5TM to generate full mtDNA sequences of more than 200 unrelated Swiss individuals living in the area of Zurich. Data were reviewed independently in two different laboratories with different software to align sequences, interpret heteroplasmy and assign haplogroups. In addition, mitogenome sequences of 50 Walser individuals were typed. The Walser inhabit alpine regions in Switzerland, Austria, as well as Italy. They are characterized by distinct linguistic and cultural features, and are considered to have remained isolated from other populations at least since the Walser migrations in the 12th and 13th centuries. We compare the haplogroup distribution of this supposedly isolated alpine population to a general Swiss population from the Zurich area, featuring a rich immigration history since the end of 19th century.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 295 P215 - CONCURRENT SEQUENCE VARIANT AT D21S11 AND Penta_D LOCI IN A PATERNITY TESTING CASE He Ren1, Zhiyong Liu2, Chen Man3, Jing Zhao4, Feng Cheng5, Chen Li6, Chuguang Chen7, Yacheng Liu8, Jiangwei Yan5

1 Beijing Police College, Beijing Police College- Beijing 102202- PR China, Beijing, China 2 Chinese Academy of Sciences-University of Chinese Academy of Sciences, Beijing Institute of Genomics, Beijing, China 3 Chinese Academy of Sciences- University of Chinese Academy of Sciences- Beijing 100049- PR China., Beijing Institute of Genomics, Beijing, China 4 Chinese Academy of Sciences- Beijing- China, Beijing Institute of Genomics, Beijing, China 5 Shanxi Medical University- Taiyuan 030001- China, School of Forensic Medicine, Taiyuan, China 6 Beijing Microread Genetics Co.- Ltd- 100089 Beijing- People’s Republic of China., Beijing Microread Genetics Co.- Ltd, Beijing, China 7 Beijing Microread Genetics Co.- Ltd- 100090 Beijing- People’s Republic of China., Beijing Microread Genetics Co.- Ltd, Beijing, China 8 Beijing Tongda Shoucheng Institute of Forensic Science- Beijing 100192- PR China., Beijing Tongda Shoucheng Institute of Forensic Science, Beijing, China

In a case of paternity testing, the 39 autosomal short tandem repeats (STRs) system reveal D21S11 and Penta_D loci from child’s chromosome 21 are unmatched with father simultaneously. Five other STR kits (PowerPlex®21, Goldeneye® 17X, AGCU 17+1, Microreader™ 23sp ID System and Microreader™ 21sp ID System) are utilized to review the genotypes of parents and child. The result shows that genotypes of X chromosome and others autosomal STR loci accord with the Mendel’s law, excepting for Penta_D and D21S11. Clone sequencing was further performed to reveal the underlying reason of these mutations. The sequence results showed that a two-step mutations and 6 nucleotides insertion (TATCTA) nearby repeat region are founded in Penta_D and D21S11 respectively. Although, it’s a rare phenomenon that two mutations are happened in the same chromosome concurrently. But once encounterd, various kits and sequencing technology contribute to finding the truth for accurate conclusion.

296 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P216 - DEEPENING IN THE Y-CHROMOSOME GENETIC LANDSCAPE OF A MICRONESIAN POPULATION Miriam Baeta1, Patricia Villaescusa1, Leire Palencia-Madrid1, Tamara Kleinbielen1, Eva Granizo1, Marian M. De Pancorbo1, Rene J Herrera2

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 Colorado College, Molecular Biology, Colorado Springs, USA

Micronesia is one of the three main zones in the Pacific Ocean, together with Polynesia and Mel- anesia. It comprises more than 2,000 islands with a land area of around 2,800 m2. The settlement of this area of Oceania represents one of the last major prehistoric human migrations. Due to its geographical location in the middle of the prehistoric Austronesian routes to the most remotes areas of Oceania, the evolutionary history of this area has long been regarded as a complex puzzle. Previous studies based on Y-chromosome in some Micronesian Islands revealed that most male lineages appear to originate from different source populations of Melanesia and East- ern Indonesia. Nonetheless, deeper studies on Y-chromosome phylogeny are needed to unveil the genetic landscape of this area. In this study, the Y chromosome lineages of the Micronesian population of Tarawa, the capital of the Republic of Kiribati, have been determined to dissect the male genetic pool of this island. In this preliminary study, a total of 96 individuals were analyzed for 23 Y-STRs using the PowerPlex® Y23 system (Promega) and haplogroup-defining Y-SNPs through a SNaPshot multiplex and/or High Resolution Melting. Haplotypes and haplogroups frequencies were determined, as well as, diversity parameters. The results revealed the contribution of paternal lineages from different ancestries, in male genetic pool of the Tarawa population, being predominant the haplogroup O, which is characteristic of Southeast Asia and Austronesian regions.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 297 P217 - DEVELOPMENT OF A NEW 17 Y-STRS SYSTEM USING FLUORESCENT-LABELLED UNIVERSAL PRIMERS AND ITS APPLICATION IN SHANXI POPULATION IN CHINA Jinding Liu1, Jiangling Guo1, Xiaojuan Cheng1, Ting Hao1, Keming Yun1, Jiangwei Yan1, Gengqian Zhang1

1 Shanxi Medical University, School of forensic medicine, Jinzhong, China

We developed a new cost-effective system of 17 Y-chromosome short tandem repeat (Y-STR) markers, which was a complement to the current commercially available Y-STR kits. It was achieved through a three primer PCR approach, involving a fluorescently labelled universal (M13–47) primer in combination with modified locus-specific primers with 5’ universal primer sequence tails. This system was divided into four panels (FAM-labelled panel DYS715, DYS709, DYS716, DYS713 and DYS607. JOE-labelled panel DYS718, DYS723, DYS708 and DYS714. TRA- MA-labelled panel DYS712, DYS717, DYS721 and DYS605. ROX-labelled panel DYS719, DYS726, DYS598 and DYS722). Target genomic fragment sizes for 17 Y-STRs were 126bp-400bp. We examined the allele and haplotype frequencies of 17 Y-STRs in 312 individuals in Shanxi province, China. 311 distinct haplotypes were obtained, and the discrimination capacity (DC) was 0.9967. The gene diversity (GD) observed across loci ranged from 0.456 (DYS598) to 0.914 (DYS712). Complete profiles were successfully amplified with as little as 0.1ng of DNA without being affected by female samples or other species. In summary, the results of our study indicate that the 17 Y-STRs have a high level of polymorphism in Chinese population and could be a robust, sensitive and cost-effective genotyping tool for forensic application and population genetic studies.

298 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P218 - DEVELOPMENTAL VALIDATION OF THE MICROREADER™ 20A ID SYSTEM Shengqiu Qu1, Yifan Li2, Chuguang Chen2, Hang Li3, Jing Zhu1, Yinji Wang1, Lin Zhang1, Weibo Liang1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Beijing Microread Genetics Co- Ltd, Beijing Microread Genetics Co- Ltd, Beijing, China 3 Yueyang Public Security Bureau, Criminal Investigation Team, Yueyang, China

The Microreader™ 20A ID system (Beijing Microread Genetics Co, Ltd, Beijing, China) is a kit designed for forensic DNA casework with superior performance and economics, which including 13 CODIS STR loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11), 3 new CODIS STR loci (D12S391, D19S433, D2S1338), 3 non-CODIS STR loci (D6S1043, Penta D, Penta E) and the amelogenin locus in one reaction with a 6-dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. The kit contains CODIS STR loci making it compatible with databases established by many public security bureaus. In our study, this system was validated according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for Forensic DNA Analysis Methods and Chinese criteria, including PCR-based studies, sensitivity study, precision and accuracy evaluation, stutter calculation, inhibitor tests, species specificity and DNA mixture studies. Our results suggested that Microreader™ 20A ID system is a useful tool for personal identification and parentage testing.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 299 P219 - DEVELOPMENTAL VALIDATION OF THE MICROREADER™ 40Y ID SYSTEM PCR AMPLIFICATION KIT Yuqing Liu1, Yifan Li2, Chuguang Chen2, Yu Tan1, Hui Jian1, Ranran Zhang1, Li Wang3, Lin Zhang1, Weibo Liang1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Beijing Microread Genetics Co- Ltd, Beijing Microread Genetics Co- Ltd, Beijing, China 3 Sichuan University, Department of Obstetrics and Gynecology, West China Second University Hospital, Chengdu, China

Y-chromosome specific STRs are commonly analyzed in forensic science, such as paternity tex- ting, familial search, especially in sexual assault cases where the identification of male DNA identity in mixed spot with high background female DNA content [1]. Microreader™ 40Y ID System, a 6-dye multiplex kit, containing 17 Y-STR loci commonly available in Yfiler kit (loci) together with other 23 Y-STR loci, is more efficient, compatible and accurate. The Microreader™ 40Y ID System (Beijing Microread Genetics Co., Ltd, China) can directly amplifies blood or saliva from a filter paper or FTA card, without template extraction and purification, and is also suitable for extracted DNA templates. Here, the validation of the Microreader™ 40Y ID System was conducted from the following aspects: sensitivity studies, amplification systems, male-male mixtures and male-female mixtures, PCR inhibitor studies, species-specificity, reproducibility, and effects of UV degradation. It provides higher personal identification capability for Y database applications. We also evaluated 400 unrelated individuals of Chinese Tibetan, Yi, Han and Uygur population, where a level of genetic sub-structure might be observed. In particular, the study of Tibetan and Yi nationality is of great help to the study of Tibetan-Yi corridor [2]. [1] Josephine Purps, Maria Geppert, Marion Nagy, Lutz Roewer, Validation of a combined autosomal/Y-chromosomal STR approach for analyzing typical biological stains in sexual-assault cases, Forensic Sci. Int. 19 (2015) 238–242. [2] Hong‐Bing Yao, Senwei Tang, Xiaotian Yao, Hui‐Yuan Yeh, The genetic admixture in Tibetan‐Yi Corridor, America Journal Of Physical Anthropology, 2017, volume 164, Issue 3.

300 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P220 - DIACHRONIC STUDY OF THE LONG TIME OCCUPIED ARCHAEOLOGICAL SITE OF SEGÓBRIGA (SPAIN) AND COMPARISON WITH NOWADAYS POPULATION Sara Palomo-Díez1,2, Rosario Cebrián-Fernández3, Ignacio Hortelano-Uceda4, Cláudia Gomes1,2, Eduardo Arroyo-Pardo1,2

Mitochondrial DNA (mtDNA) analysis is an important source of information about biogeographical mother lineage origin of the individuals. Furthermore, when we face up critical DNA samples, many times mtDNA is the only source of genetic information. Even, sometimes, this genetic marker could provide information about kinship through maternal lineage. This study is focused on 14 archaeological individuals from the Segóbriga archaeological site (Saélices, Cuenca, central-eastern Spain). The relevance of Segóbriga is that it has been occupied by different human populations throughout ten centuries; from the Roman Imperial period of the Iberian Peninsula, until the end of the Muslim period. So that, the 14 selected individuals belong to different periods within the total time of occupation of the site; concretely there were selected: five from the Muslim period, seven from Late Roman period, one Visigoth and one individual without a precise chronology assigned. We have studied the HVI and HVII mitochondrial DNA regions, by the amplification of short overlapping fragments. The objectives of this analysis were: the search of the biogeographical origin of the individuals, and the establishment of comparisons among the different chronological groups, including nowadays population of the region, trying to evaluate the genetic contribution of the past populations in the current population. We have obtained HVI and HVII regions mtDNA profiles in 8 of the 14 individuals, establishing their mtDNA haplogroup; and only partial HVII region mitochondrial DNA profiles in 5 of them. All the individuals shown typical European mtDNA haplogroups (H2a2a1g, H5, H5a, K, U5a, U5b1c, T2e, H), some of them with significant nowadays presence in Middle East (T2e, U5b1c, U5a and H5).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 301 P221 - DNA IDENTIFICATION OF SKELETAL REMAINS BY INVESTIGATOR’S INTUITION Ugo Ricci1, Claudia Centrone2, Francesca Gerundino3, Elisabetta Pelo3

1 AOU Careggi SOD Diagnostica Genetica, Forensic Genetics Unit, Florence, Italy 2 Azienda Ospedaliero Universitaria Careggi, SOD Diagnostica Genetica Forensic Genetic Unit, Florence, Italy 3 Azienda Ospedaliero Universitaria Careggi, SOD Diagnostica Genetica, Florence, Italy

In June 2006 a decapitated woman was found in a parking area of the motorway in the area of Prato (Florence). Since the body was beheaded and no victim’s documents or objects were present at the crime scene, identification at that time was impossible. However, DNA profile from woman’s bones were collected. In the same year a mother had reported her daughter’s disappearance, but the two events were not related at that time. About ten years later the mother’s DNA profile was finally acquired for a genetic identification of another girl’s body found in the Ferrara area. These genetic profiles were completely discordant. All these genetic comparisons were carried out on behalf of the prosecutors of the cities involved in the findings of the bodies and in the disappearance complaints, but due to the lack of a database the events remained disconnected. In January 2017, the head of the scientific police of Prato who had followed the investigation and questioned the mother of the missing girl found about ten years later, suggested to the magis- trate to order the comparison of the mother’s DNA with the genetic profile of the bones found in 2006. This comparison finally allowed the identification of the missing daughter. Databases of unknown bodies and relatives of missing persons were created in Italy as a part of national DNA database just at the beginning of 2018. This report highlights the importance of having forensic DNA Database to search for missing persons and how the investigator’s intuition can play a key role in resolving criminal cases.

302 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P222 - ESTIMATES ANCESTRY PROPORTIONS OF THE MIDWEST REGION (BRAZIL) Fernanda Silva Polverari1, Danilo Faustino Braganholi1, Isabela Brunelli Ambrosio1, Juliana Martinez1, Bianca Belon Januário1, Flávia Alves de Jesus Silva1, Regina Maria Barretto Cicarelli1

1 Faculdade de Ciências Farmacêuticas - UNESP, Ciências Biológicas, Araraquara-SP, Brazil

Brazilian population history formation indicates an admixed between europeans, africans and native americans, thus midwest region population presents these three ancestral groups due to migration movements. Currently, this country region is politically divided by the states of Mato Grosso, Mato Grosso do Sul and Goiás, and is also where Brasilia DC is located. In this study, proportions of population ancestry were verified by the analysis of 32 X-INDELs compared to data from autosomic markers, mtDNA and Y chromosome. For this, natural unrelated individuals samples from Mato Grosso State and Brasília were analized with X-INDELs. Ancestry proportions identified for Mato Grosso population were: 34 % EUR, 34 % AFR and 32 % NAM, and, for Brasília population: 38 % EUR, 35 % AFR and 26 % NAM. Autosomic markres studies indicated a majority European ancestry (59 %, 26 % AFR and 15 % NAM). When uniparental markers are evaluated it is possible to verify interesting particularities between male and female ancestral lineages. A recent study indicated african Ancestry prevalence from mtDNA (49 % AFR, 33 % NAM and 18 % EUR), while European ancestry is much higher Y analysis (90 % EUR, 8.5 % AFR and 1.5 % NAM). In this context, in which the region population admixed is large and variable between lineages, X chromosome bias to reflect a balanced contribution between Europeans, Africans and Native Americans.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 303 P223 - ESTIMATING THE INFORMATIVENESS OF FORENSIC ANCESTRY MARKERS Elaine Y.Y. Cheung1, Christopher Phillips2, Mayra Eduardoff3, María Victoria Lareu2, Dennis McNevin4

1 National Centre for Forensic Studies, University of Canberra, Canberra, Australia 2 Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 4 Centre for Forensic Science, University of Technology Sydney, Sydney, Australia

The forensic inference of biogeographical ancestry (BGA) applying the analysis of bi-allelic and tri-allelic single nucleotide polymorphisms (SNPs) has been highly successful. More recently, microhaplotypes (MHs) have emerged as a new forensic marker capable of both mixture deconvolution and ancestry inference. With the candidate pool of ancestry-informative markers (AIMs) growing, we have developed a novel marker selection procedure that sequentially adds AIMs to a forensic panel while ensuring a balance of population-specific markers. Well-established population divergence metrics were used to rank SNP and MH markers by their informativeness (or differentiation potential) for five reference populations from the 1000 Genomes database. STRUCTURE [1] analyses were then performed to determine the level of differentiation between African, European, South Asian, East Asian, and American population groups. This study demonstrates that individual metrics can be biased towards the selection of markers with particular characteristics. For example, FST favours loci with large allele frequency differences between populations and low heterozygosity, but this metric does not properly take into account allelic diversity. As a result, FST over-estimated the differentiation potential of bi-allelic SNPs that satisfy this condition, despite a panel of MHs giving better ancestry inference performance than the top ranked SNPs. Overall, populations were well differentiated with panels consisting of both SNPs and MHs, however, comparison of individual markers revealed a majority of MHs outperform bi-allelic and tri-allelic SNPs. 1. Pritchard J.K., et al: Inference of population structure using multilocus genotype data, Genet- ics. 2000; 155: 945–959.

304 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P224 - EVALUATING THE IMPACT OF THE Y CHROMOSOME HAPLOGROUP R1B-DF27 IN HISPANIC ADMIXED POPULATIONS FROM LATIN AMERICA Patricia Villaescusa1, Paula Blázquez-Martín1, Begoña Martínez-Jarreta2, Susana Jiménez3, Ana María Rocandio1, Marian M. De Pancorbo1

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 University of Zaragoza, Research Group GIIS-063 IIS-Aragón and Department of Forensic Medicine and Toxicology, Zaragoza, Spain 3 University Miguel Hernandez Elche, Forensic Medicine Division, Department of Pathology and Surgery, Alicante, Spain

The populations of Latin America have been influenced historically by male-mediated migrations during the colonial period stemming from Spain and Portugal. The admixture of the local populations of Native Americans and Europeans gave rise to the admixed populations known as Mestizos, characterized by a European-Native American mixed ancestry where the paternal lineage is usually of European origin. The analysis of Y chromosome SNPs allows us to stablish haplogroups and paternal biogeographical ancestry. R1b-M269, and its subhaploproup S116, are among most common European haplogroups. The S116 derived lineage DF27 has been found be near-specific of the Iberian Peninsula and its analysis could contribute to deepen the paternal the European/Iberian introgression in Latin America. The objective of this work is to study the presence of DF27 in five Mestizo populations from Panama, Nicaragua, El Salvador, Guatemala and Colombia. In order to do that we analyzed the Y-SNP DF27 in 230 individuals and in addition, we collected population data from Latin American populations from the 1000 Genomes database. The obtained results reveal an average frequency of 29–35 % in the analyzed populations, being in concordance with the frequencies of DF27 observed in the 1000 Genomes database. Furthermore, the distribution of the frequencies observed in the five analyzed populations seem to agree with the influence of the main trade routes between Spain and America in that area during the colonial era, which allows us to explain the possible reason behind the higher frequencies found in Colombia, where the main port linked to Spanish trade was located.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 305 P225 - EVALUATION OF 30 INSERTION/DELETION POLYMORPHISMS AS FORENSIC MARKERS IN THE KUWAITI POPULATION Mahdi Haidar1,2, Hussain Alsaleh1,2, Penelope R. Haddrill1

1 University of Strathclyde, Centre for Forensic Science, Department of Pure and Applied Chemistry, Glasgow- Scotland, United Kingdom 2 Kuwait Identification DNA Laboratory, General Department of Criminal Evidence, Ministry of Interior, Kuwait City, Kuwait

This study evaluates the forensic utility of the 30 insertion and deletion (indel) markers contained in the Qiagen Investigator® DIPplex kit in the Kuwaiti population. Allele frequencies and forensic indices were studied in 150 unrelated Kuwaiti individuals. All but one of the 30 markers were shown to conform to the expectations of the Hardy-Weinberg Equilibrium. Linkage disequilibrium tests for all possible pairwise combinations of loci showed no statistically significant deviation from independence. The genotyping results showed that the DIPplex markers display artefacts, which were most likely due to the presence of genetic variants in the primer binding region. The high combined power of discrimination (CPD) of more than 99.999 % and low combined match probability (CMP) of 2.736 x 10–13 provide a satisfactory level of discrimination, allowing the DIPplex loci to be used as forensic markers for individual identification in Kuwait. The typical paternity index (TPI) ranged between 0.075 to 1.13 and the combined power of exclusion (CPE) calculated from the 30 markers was 99.46 % indicating the usefulness of the DIPplex kit as a supplementary typing system for challenging paternity/kinship cases in Kuwait. Finally, when principal component analysis (PCA) and STRUCTURE were implemented on the DIPplex marker dataset, no genetic structure was detected within the Kuwaiti population. However, PCA revealed a correlation between geographic and genetic distance, when allele frequencies from other reference populations were used.

306 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P226 - EVALUATION OF HOMOPLASY IN Y23 LOCI: A PILOT STUDY ON SAMPLES WITH KNOWN 400-YEARS-LONG GENEALOGICAL HISTORY IN EASTERN CZECHIA Vlastimil Stenzl1, Radim Vasut2

1 Institute of Criminalistic, Genetics, Prague, Czech Republic 2 Palacky University in Olomouc, Botany, Prague, Czech Republic

Surname genealogies co-evolve with Y-STR profile in European societies and generally can infer from each other. Close similarities in matrices of 23 Y-STR loci are considered to be related to genetic relationship between persons. With increasing numbers of tested persons in Mora- via (1000+ individuals), we are detecting increasing number of similar Y23 profiles in lineages with known genealogies (usually pedigrees of 300–400 yr old). Such similarities that originate in the same geographic region might reflect past genetic relationship (before year 1600). In order to evaluate events of homoplasy in such similarities in Y23 profiles, we sequenced (Illumina MiS- eq) profiles of 2 surnames, which are known to be genetically related (in past 400 years). The occurrence, rate and chance of misinterpretation due to homoplasy is discussed on example of genealogies of the east-Moravian clans.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 307 P227 - EVALUATION OF RAPIDLY MUTATING Y-STRS IN PAKISTANI POPULATION Muhammad Qari Absar Ali1, Rashed Alghafri2,3, Muhammad Farhatullah1, Reem Almheiri2, Ahmed Adam2, Muhammad Ramzan Khan1

1 Quaid-i-Azam University, Biotechnology, Islamabad, Pakistan 2 Dubai Police GHQ, General Department of Forensic Science and Criminology, Dubai, United Arab Emirates 3 United Arab Emirates University, Biology, Al-Ain, United Arab Emirates

Y-chromosomal Short Tandem Repeats (Y-STRs) have been widely used in forensic investigations and population genetics. Commercially available Y-STRs kits allow the identification of male pedigrees and limited application in forensic genetics because of its limitation in differentiating related male individuals. Recent research with the Rapidly Mutating Y-STRs (RM Y-STRs) has revealed that these loci furnish significantly higher haplotype discrimination capacity and haplotype diversity in worldwide populations when compared with the conventional Y-STRs applied in forensic genetics. Although the number of RM Y-s loci has found its way in most updated commercial kits, there are still some loci not yet used in such kits. The aim of this study is to develop RM Y-STR haplotypes frequency database for Pakistan population, in order to appraise the resolution power of these loci. Blood samples from unrelated male individuals were collected in vacutainers. Amplification of RM Y-STR was done using RM-Yplex assay which comprises 13 loci including, DYF387S1, DYF399S1, DYF403S1a/b, DYF404S1, DYS449, DYS518, DYS526a/b, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627. Forensic parameters including, discrimination capacity (DC), haplotype diversity (HD), gene diversity (GD) and random match probability (RMP), were estimated in this study. All haplotypes were searched against previously published haplotypes databases for the same markers in order to identify shared haplotypes among and within worldwide populations.

308 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P228 - FALSE POSITIVE RESULTS IN PATERNITY TESTINGS BASED ON NUMBER OF ANALYZED LOCI Dejan Šorgić1, Dijana Takić Miladinov1, Aleksandra Stefanović1

1 Institute of Legal Medicine, DNA laboratory, Niš, Serbia

The aim of the present study was to evaluate the importance of including the greater number of loci in paternity tests - particularly in cases when mother is not involved - for the purpose of minimising the risk of false positive results. It is a well-known fact that DNA testing based on the STR analysis is the most accurate method when detemining if someone is a biological father of the child. Although false positive results can occur if father or brother of alleged father are tested, we wanted to determine if false positive results can possibly be due to a random match of alleles between unrelated individuals in cases when mother is excluded from DNA testing and there is no knowlege of alleles inherited from her. The DNA profiles of 300 children were analysed and consequently compared to 1200 unrelated individuals. All DNA profiles used were part of our database, and as part of our regular procedure informed written consent was previously obtaind for each of them, allowing the data to be used anonymously for research purposes. In the first stage, the analyses were performed for 15 STR loci (D3S1358, D1S1656, D2S441, D10S1248, D16S539, D18S51, D2S1338, TH01, vWA, D21S11, D8S1179, D12S391, D19S433, FGA, D22S1045), whereas in the second stage, all 300 DNA profiles were reanalysed with 8 additional STR loci (D13S317, Penta E, CSF1PO, Penta D, D7S820, D5S818, TPOX, DYS391), which amounts to 23 loci in total. The results of this study revealed a significant decrease in the number of false positive results in the group with the additional 8 loci. Key words: paternity tests, false positive results, STR analysis, number of loci.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 309 P229 - FIRST-DEGREE FAMILIAL RELATIONSHIPS COINCIDENCES IN A POPULATION DATABASE OF JUJUY (ARGENTINA) COMPARED WITH SIMULATED POPULATIONS Juan José Martínez1, Marcos Felipe Tula Molina2, Luis Adrián Sánchez2, María Inés Deus2, María Isabel Ramella2, Gladys Beatriz Maurín2, Gustavo Martínez3, Ariel Chernomoretz4,5,6, María Soledad Escobar7, Gustavo Sibilla7,María Cecilia Miozzo2

1 Instituto de Ecorregiones Andinas, CONICET- Universidad Nacional de Jujuy, San Salvador de Jujuy, Argentina 2 Laboratorio Regional de Genética Forense del NOA, Departamento Médico - Poder Judicial de Jujuy, San Salvador de Jujuy, Argentina 3 Servicio de Genética Forense y Registro Provincial de Datos Genéticos, Superior Tribunal de Justicia de la Provincia de Entre Ríos, Paraná, Argentina 4 Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Buenos Aires, Buenos Aires, Argentina 5 Instituto de Física de Buenos Aires IFIBA, Universidad Nacional de Buenos Aires- CONICET, Buenos Aires, Argentina 6 Laboratorio de Biología de Sistemas Integrativa, Fundación Instituto Leloir, Buenos Aires, Argentina 7 Fundación Sadosky, Secretaría de Ciencia- Tecnología e Innovación Productiva, Buenos Aires, Argentina

Jujuy, a province from Northwestern Argentina, has a population with great input of Native American gene pool, together with European and African contribution. For forensic purposes a frequency population database was built selecting 500 non related individuals from Jujuy province and genotyping them with 21 autosomal STRs from the kit GlobalFiler. The genetic profiles were incorporated in GENis (a software developed in Argentina for storage and comparison of DNA profiles) to corroborate there were no matches in high stringency. After this confirmation, we run a searching strategy with low stringency, which is used to find parent-child relationships. Four (4) adventitious matches were found. To investigate if this result was due to a specific population characteristic of Jujuy, 100 populations, with 500 random genetic profiles each one, were simulated using Jujuy allele frequencies. For this purpose the forensim R package was used and every population was analyzed with GENis with low stringency. The coincidences for these populations were in average 1.74 (95 % CI: 1.5–2), even though 4 matches were found in 5 populations, 5 matches in 2 and there was one population with 6 matches. There were no significant differences between Jujuy and the simulated populations, so we disregard that adventitious matches are because of a specific Jujuy population characteristic. It is known that the number of adventitious matches increases when you go down with stringency in DNA databases, but analyzing a high number of autosomal STRs, the 4 matches were unexpected. So, care should be taken with first-degree relationships searches even with high number of markers. LR calculations and complementary studies (like Y chromosome and mitochondrial DNA) must be done in this kind of searches.

310 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P230 - FORENSIC AND POPULATION GENETIC ANALYSIS OF SERBIAN POPULATION USING 21 STR LOCI OF GLOBALFILER™ PCR AMPLIFICATION KIT Dragana Zgonjanin1, Reem Almheiri2, Rashed Alghafri2, Dalibor Nedić3, Goran Stojiljković1

1 Clinical Center of Vojvodina, Institute of Forensic Medicine, Novi Sad, Serbia 2 Dubai Police G.H.Q., General Department of Forensic Sciences and Criminology, Dubai, United Arab Emirates 3 Clinical Center of Banja Luka, I Institute of Forensic Medicine of Republic of Srpska, Banja Luka, Bosnia and Herzegovina

Autosomal short tandem repeats (STRs) have been widely used in forensic investigations. Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. The 6-dye GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) comprises 21 autosomal STRs have already been proven to be able to provide reliable DNA profiling results and enhance the power of discrimination between individuals. In this study, we are presenting an analysis of GlobalFiler STR loci on 209 unrelated individuals from Serbia. Samples were collected using buccal swabs and extracted using PrepFiler® Extraction method. Amplification was conducted as per manufacturer protocol and samples were run on ABI 3500 Genetic Analyzer. No significant deviations from Hardy– Weinberg equilibrium and linkage disequilibrium were detected within and between the 21 STR loci. SE33 has shown the greatest power of discrimination in Serbia population, whereas TPOX has shown the lowest. The combined power of discrimination was found to be 99.99999999999 9999999999989954 %. AMOVA test was conducted with the relevant populations in the region using Arlequin software v3.5. Power of discrimination, gene diversity and alleles frequencies presented in this study suggest that the 21 STR loci have great value in forensic casework analysis as well as in kinship analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 311 P231 - FORENSIC CHARACTERISTICS OF TIBETO-BURMAN- SPEAKING TIBETANS REVEALED BY 50 INDELS Yiping Hou1, Mengge Wang1, Zheng Wang1, Guanglin He1

1 Sichuan University, Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Chengdu, China

Insertion/deletion polymorphism (InDel) is a promising tool in forensic investigations and biogeographical ancestry inferences. Recently, an novel InDel kit has been designed by the AGCU company and allows co-amplification and fluorescent detection of the 47 autosomal InDels (A-InDels), 2 Y-chromosomal InDels (Y-InDels) and Amelogenin. In this study, 103 Tibeto-Bur- man-speaking Tibetan individuals were genotyped to investigate the effectiveness of this novel InDel kit. No significant deviation from Hardy-Weinberg equilibrium was observed at the 47 A-InDels after applying the Bonferroni correction. The allelic frequencies of insertion alleles at the 47 A-InDels ranged from 0.2087 to 0.7767, and the observed heterozygosity varied from 0.3010 to 0.5922. The combined power of discrimination was 1 - 4.0688E-19 and the combined probability of exclusion was 0.9997. Our results demonstrate that this novel InDel kit can be used as an efficient tool for individual identification and forensic paternity testing.

312 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P232 - FORENSIC FEATURES OF CHINESE HAN AND GENETIC STRUCTURE AT INSERTION/DELETION POLYMORPHISMS WITH OTHER 31 CHINESE ETHNIC GROUPS Yiping Hou1, Xing Zou1, Zheng Wang1, Guanglin He1, Hong Zhu1

1 Sichuan University, Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Chengdu, China

FORENSIC FEATURES AND GENETIC STRUCTURE AT INSERTION/DELETION POLYMORPHISMS OF CHINESE HAN WITH OTHER 31 CHINESE ETHNIC GROUPS Xing Zou1, Zheng Wang1, Guanglin He1, Hong Zhu1, Yiping Hou1

1 Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Sichuan University, Chengdu 610041, China

Insertion/deletion polymorphisms (InDels) have been used in population genetics, anthropology, archaeology and forensic. Chinese Han population with 1.3 billion people are the largest homogeneous ethnicity in the world. Here, we reanalyzed the allele frequency distributions and forensic parameters of 30 InDels included in Qiagen Investigator DIPplex kit in a Meta-Han Chi- nese population and then explored the genetic affinity between Meta-Han group and other 31 Chinese ethnic populations belonging to different language families using Fst genetic distances, principal component analysis (PCA), multidimensional scaling (MDS), phylogenetic analysis and Structure. The values of CPD and CPE in Han Chinese are 0.99999999998532 and 0.9868, respectively, which indicating the panel is informative for forensic individual identification, while just be served as a supplementary tool for parentage testing. The comprehensive population comparisons reveal that Han Chinese have the genetically closer relationship with Hmong- Mien-speaking groups, such as Fujian She and Zunyi Miao, than Tibetan-Burman-speaking and Turkic-speaking groups. Findings from pairwise genetic distances, PCA, MDS and phylogenetic analysis demonstrate that genetic affinity is not only correlated with ethnic origin, geographical distribution, but also with linguistic origin. STRUCTURE analysis suggests that the same language origin populations have similar ancestry composition.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 313 P233 - FORENSIC GENETIC ANALYSIS OF SOUTH PORTUGUESE POPULATION WITH THE SIX DYE POWERPLEX® FUSION 6C Cláudia Vieira Da Silva1, Heloísa Afonso Costa1,2, Maria João Porto1, Eugénia Cunha3,4, Francisco Corte Real3,5, António Amorim1,6

1 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Serviço de Genética e Biologia Forenses, Lisboa, Portugal 2 Universidade da Beira Interior, Faculdade de Ciências da Saúde, Covilhã, Portugal 3 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Instituto Nacional de Medicina Legal, Lisboa, Portugal 4 Universidade de Coimbra, Faculdade de Ciências e Tecnologia, Coimbra, Portugal 5 Universidade de Coimbra, Faculdade de Medicina, Coimbra, Portugal 6 Universidade de Lisboa, Faculdade de Ciências, Lisboa, Portugal

As an improvement in efficiency and in Human Discrimination Power, the new six dye multiplex kit PowerPlex® Fusion 6C System, by Promega, available for human identification can co-amplify 27 loci, in a single reaction, have been introduced in the last years with great success. This kit allows the amplification and detection of autosomal loci included in the expanded Combined DNA Index System CODIS, plus the loci Penta D, PENTA E and SE33 as well as Amelogenin for gender determination. Furthermore, this kit includes three Y –STRs (DYS391, DYS576, and DYS570), allowing allelic attribution in a total of 27 loci. This genetic markers extension satisfies not only CODIS but also European Standard Set recommendations. Thinking about continuous human migration movements, especially in a very cosmopolitan region like Lisbon and south of Portugal, and also, in keeping population studies and actualized STR databases we decided to update our previous studies. Our sample is composed of 600 unrelated individuals, from paternity testing with laboratory identity anonymized. DNA was extracted by Prep-n-go BufferTM(Thermo-Fisher Scientific). PCR amplification was performed with PowerPlex® Fusion 6C System, according to the manufacturer’s guidelines. Fragment analysis was carried out in an Applied Biosystems® 3500 Genetic Analyser. Electrophoresis results were analyzed with GeneMapper® ID-X v1.4. Allele frequencies and population statistics, including Hardy-Weinberg equilibrium p-values from exact test probabilities and forensic parameters, were calculated with adequate software. In conclusion, our population information was updated in order to apply the most recent data in our casework weight of evidence.

314 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P234 - FORENSIC GENETIC ANALYSIS OF THE POPULATION OF GUJARAT WITH POWERPLEX 21 MULTIPLEX SYSTEM Aditi Mishra1, Sumit Kumar Choudhary1, Pankaj Shrivastava2

1 Raksha Shakti University, Department of Forensic Science, Ahmedabad, India 2 State Forensic Science Laboratory, DNA Fingerprinting Unit, Bhopal, India

The present study evaluated the allele frequencies and forensic parameters for the loci included in the PowerPlex 21 multiplex system in 101 unrelated individuals residing in the state of Gujarat, India. The study also presents the first global report on polymorphism on Penta D and Penta E autosomal STR loci from the population of Gujarat, India. The obtained results revealed that the studied STR multiplex system is highly polymorphic and suitable for forensic genetic analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 315 P235 - FORENSIC PARAMETERS AND GENETIC STRUCTURE BASED ON Y-CHROMOSOME SHORT TANDEM REPEATS IN LESOTHO POPULATION Motsoboeana Mpasi Lesaoana1,2, Mohaimin Kasu1, Maria Eugenia D’Amato1

1 University of the Western Cape, Biotechnology, Western Cape, South Africa 2 Lesotho Mounted Police Service, Forensic Laboratory, Maseru, Lesotho

In the early 1800’s during Lifaqane wars, a significant numbers of Nguni tribes (Ndebele, Xhosa; and Vundle) migrated into Lesotho where they were insulated by the autochthonous Sotho, Phuti (also Nguni tribe) and aboriginal San bushmen. Lesotho population of ~ 2.2 million people is dominated by the Southern Sotho ethnic group (~97 %).We evaluated the genetic diversity and the discrimination capacity (DC) of the UniQ-Typer Y-10TM in 939 unrelated males from these five Bantu groups (Southern Sotho = 620, Ndebele = 120, Xhosa = 110, Phuthi = 50, and Vun- dle = 39) with UniQTyper genotyping system. We observed 588 singletons and 111 haplotypes shared among 351 individuals. The overall haplotype diversity (HD) and discrimination capacity (DC) were 0.9264 and 0.744 respectively. AMOVA analysis excluding individuals with duplications and nulls indicated the presence of structure among the 5 populations (Va = 5.35 %). An MDS plot revealed a close genetic relationship between the Sotho, Ndebele and Phuthi who clustered away from the Xhosa and Vundle. We concluded that the absorption of ethnolinguistic identity of the Ndebele and Phuthi tribes into Sotho culture was more intense than for the Xho- sa and Vundle, and that the population structure observed in Lesotho population was created by language rather than geographic distance.

316 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P236 - FORENSIC STATISTICAL PARAMETERS OF 22 AUTOSOMAL STRs IN MESTIZOS FROM THE PENÍNSULA DE YUCATÁN, MEXICO Javier Enrique Sosa-Escalante1, Lizbeth Gonzalez-Herrera2, Paola Lopez- Gonzalez1, Maria Jose Lopez‑Gonzalez1, Rosalba Yaqueline Gamboa-Magaña3, Refugio Guadalupe Herrera‑Diaz3, Karla Adriana Piña-Dzul3, Stefany Fabiola Leon-Acosta3, Rolando Ismael Flores-Baas3, Jorge Enrique Bautista-Gonzalez1, Maria Rocio Rivero-Salazar4, Hector Rangel-Villalobos5

1 DIMYGEN Laboratorio, Laboratorio Privado, Merida, Mexico 2 Universidad Autonoma de Yucatan, Laboratorio de Genetica. Centro de Investigaciones Regionales, Merida, Mexico 3 Instituto de Ciencias Forenses del Estado de Yucatan, Direccion de Quimica y Genetica Forense, Merida, Mexico 4 Genomelab Mexico, Genomelab Mexico, Tijuana, Mexico 5 Instituto de Investigación en Genetica Molecular, Ocotlan, Mexico

The mestizo (admixed) population of the Peninsula of Yucatan (Southeast, México) characterizes by a predominant Native American origin involving the Mayan culture. Autosomal Short tandem repeats (STRs) are the election tool for human identification (HID) purposes, such as forensic casework and paternity testing, among others. Although there are autosomal STR databases for some Mexican populations, the new HID kits have poorly studied in this country, such as the Powerplex Phusion system (Promega, Corp.). Therefore, we estimated their allele frequencies and forensic parameters for 22 autosomal STRs in a population sample of 687 Mexican Mesti- zos subjects from the Peninsula of Yucatan (Southeast, Mexico). Genotype distribution was in agreement with Hardy-Weinberg expectations (p>0.01), except for D1S1656, and 85 different alleles were observed in the studied population. For each locus, allele frequencies were obtained, ranging from 0.0007 to: 0.4766 for D3S1358; 0.1739 D1S1656, 0.2525 D2S441, 0.3781 D10S1248, 0.2271 D13S317, 0.1907 PENTA E, 0.2874 D16S539, 0.1963 D18S51, 0.2589 D2S1338, 0.2756 CSF- 1PO, 0.2430 PENTA D, 0.3903 THO1, 0.3960 VWA, 0.2656 D21S11, 0.2993 D7S820, 0.4993 D5S818, 0.4204 TPOX, 0.3343 D8S1179, 0.2289 D12S391, 0.2629 D19S433, 0.1585 FGA, 0.4597 D22S1045. FGA and Penta E were the most polymorphic loci with 27 and 25 different alleles, respectively. Heterozygosity ranged from 0.6405 for D2S441to 0.9076 for D1S1656.The most discriminating loci were Penta E (PD = 0.9844) and FGA (PD = 0.9756). The random matching index was 2.7207 X 1025. Our results provides and STR population dataset that allow confident interpretation of paternity tests and criminal cases carried out with this HID system in the Peninsula of Yucatan.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 317 P237 - FREQUENCY OF NULL ALLELES IN PATERNITY CASES FROM MEXICO FOR THE LOCUS PENTA E Hector Rangel-Villalobos1, José Alonso Aguilar-Velazquez1, Mayra Elizabeth García-Aceves1, Carolina de Pilar Gutiérrez-González2, Gabriela Martínez-Cortés1

1 Universidad de Guadalajara, Instituto de Investigación en Genética Molecular CUCI-UdeG, Ocotlán- Jalisco, Mexico 2 DNA Profile SC, Laboratory of Genetics, Ocotlán- Jalisco, Mexico

We report herein two peculiar motherless paternity cases received in two consecutive weeks involving three father-child mismatches for Penta E, the autosomal STR loci included in the Pow- erplex Fusion system. In the first case, based on the residual Paternity Index (PI), the kinship was confirmed between the father and three children (PI > 99.999 %). However, the father homozygous 16/16 did not match with two children homozygous 5/5 for the Penta E locus, whereas the third child was homozygous 16/16. Unexpectedly, the next week we detected another unique mismatch for Penta E between one father homozygous 9/9 and the child homozygous 17/17, and residual PI also confirmed the kinship (PI > 99.999 %). Consequently, these three paternity test mismatches were faced as null alleles, which are described as variants in the primer-binding region that can lead to allele dropout resulting as false homozygous. Interestingly, allele nulls are not described in the STRBase for Penta E (https://strbase.nist.gov/NullAlleles.htm). Thus, according with our lab’s records, we estimated a preliminary null allele frequency for Penta E in Mexico based on the number cases (6/3299; 0.182 %). Although this frequency seems very low, probably this is underrepresented because estimation comes from motherless paternity cases and require further research. In brief, this report provides helpful information for reliable interpretation of paternity cases with unique mismatches for Penta E, and helps to promote the research or modification of the corresponding primers by the supplier (Promega Corp.).

318 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P238 - GENES INVOLVED IN DAMAGE RESPONSE CAUSED BY UV RADIATION IN ECUADORIAN POPULATION OF DIFFERENT ALTITUDE REGIONS Paola E. Leone1, Patricia Guevara-Ramírez1, Ana Karina Zambrano1, Andy Pérez-Villa1, Isaac Armendáriz-Castillo1, Jennyfer M. García-Cárdenas1, Santiago Guerrero1, Andrés López-Cortés1, Verónica Yumiceba1, César Paz-y-Miño1

1 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo- Universidad UTE, Quito, Ecuador

Ecuador has various regions at different altitudes. It is known that at high altitudes, organisms experience multiple stressors, including exposure to UV radiation. Even, ultraviolet radiation exposure increases when getting closer to the equator line; indeed, UV radiation increases at 4 % every 300 meters of altitude. Consequently, cities in the inter-Andean region of Ecuador, located at about 2800–3000 meters above sea level (masl), are exposed to UV levels approximately 40 % higher than those of the lowland regions. Ultraviolet light is a carcinogen that causes damage to DNA and as a result mutations or cellular apoptosis do occur. However, these alterations can be counteracted by genes such as XPC, XPD and XPG that encode proteins which perform crucial functions in the recognition and repair of damage caused by UV radiation. The aim of the present study was to evaluate the distribution of three polymorphisms (rs2228001, rs13181 and rs17655) involved in the response to the damage caused by UV radiation in the Ecuadorian populations of high and low altitudes, and thus, correlate the ancestral proportions of these populations. Re- sult analysis showed that the behavior or adaptation of both groups located at different altitudes is similar. The ancestry of these groups exhibited that the Native American component prevails, and only the proportion of the European and African varies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 319 P239 - GENETIC ANALYSIS OF 12 X-CHROMOSOMAL STRs AN AUTOCHTHONOUS POPULATION OF SOUTHEAST SPAIN Susana Jiménez1, Celia García1, Ester Lobato1, Miriam Baeta2, Eladio Bañón1, Marian M. de Pancorbo2

1 University Miguel Hernández, Patología y Cirugía, Sant Joan d’Alacant, Spain 2 University of the Basque Country UPV/EHU, BIOMICs Research Group- Lascaray Research Center, Vitoria- Gasteiz, Spain

The analysis of X-chromosomal short tandem repeats (STRs) is a valuable tool as well as a useful complement to the study of autosomal and Y chromosome STRs in complex kinship cases due to the particular inheritance of the X chromosome. Currently it is necessary to expand the study of a greater number of X-STRs, and know their population characteristics, for better forensic purposes. The allelic frequencies and further population forensic genetic parameters were studied in 12 X-Chromosome STRs loci included in the InvestigatorTM Argus X-12 Kit. Data has been obtained from 120 unrelated autochthonous subjects, 60 females, and 60 males an area of southeast Spain, with relevant demographic and historical characteristics. Allele frequencies for each marker were determined, and the most frequent alleles observed have been: DXS10103 (allele 19 ), DXS8378 (alleles 10 and 12), DXS10101 (allele 30.2), DXS10134 (allele 36), DXS10074 (allele 18 ), DXS7132 (allele 13), DXS10135 (allele 21), DXS7423 (allele 15), DXS10146 (allele 29), DXS10079 (allele 19 ), DXSHPRTB (allele 12), and DXS10148 (alleles 24.1 and 26.1). The obtained data will contribute to further progress in the studies on chromosome X in Spain. Comparison with other Spanish populations showed some differences which were analysed.

320 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P240 - GENETIC CHARACTERIZATION AND ANCESTRY OF THE ADMIXED POPULATION OF MARAJÓ ISLAND, NORTHERN OF BRAZIL, WITH AUTOSOMAL AND LINEAGE MARKERS Silvia Martins1, Filipa Simão1, Luiz Marcelo Pinheiro2, Masinda Nguidi1, Lara Deccache1, Leonor Gusmão1, Elizeu Fagundes de Carvalho1

1 State University of Rio de Janeiro, Laboratório de Diagnósticos por DNA, Rio de Janeiro, Brazil 2 Federal University of Pará, Department of Natural Sciences, Soure, Brazil

The Island of Marajó is in the north of Pará state, Brazil. At the time of Portuguese colonization, it is believed that 30 different indigenous groups inhabited the Island. Currently it is composed by an admixed population, as a result of interethnic mating between European colonizers, Native Americans and African slaves. To investigate the genetic background of the Marajó population, a sample of unrelated individuals was selected, with matrilineal or patrilineal local ancestry for at least three generations. A total of 72 males were genotyped for the 27 Y chromosome STR included in the YFiler Plus kit. Additionally, 55 samples were sequenced for the complete mtDNA control region and 46 were genotyped for 46 autosomal insertion-deletion ancestry informative markers, in a single multiplex reaction. The haplotype diversities obtained for Y-chromosome and mtDNA markers are lower (0.9969±0.0032 and 0.9833±0.0097, respectively) when compared with other Brazilian admixed populations, probably due to the geographic isolation of the island. In the comparison with data available for HVI, significant FST genetic distances were found between Marajó and samples from other regions of Brazil. The mtDNA haplogroup composition revealed a high contribution of Native American maternal ancestry of approximately 66 %, followed by 29 % of African haplogroups and only 5 % where from European origin. In the study of the autosomal ancestry of the individuals, a 42.8 % of European ancestry was found, followed by 30.9 % of Native American and 26.3 % of African contributions. These markers show high variation throughout Brazil and the results found are in agreement with the literature available for the North region.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 321 P241 - GENETIC CHARACTERIZATION OF 12 STRS (INVESTIGATOR HDPLEX) IN ECUADORIAN POPULATION Anibal Gaviria1, Margarita Vela1, Gisella Fiallos1, Carmen Gruezo1, Cristina Rodríguez1, Juan José Builes2, Ana Karina Zambrano3

1 Laboratorio de Genética, Centros Médicos Especializados Cruz Roja Ecuatoriana, Quito, Ecuador 2 GENES Ltda, Laboratorio de Genética Forense y Huellas digitales del DNA, Medellin, Colombia 3 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo-Universidad UTE, Quito, Ecuador

Investigator HDplex kit includes 12 STR markers and Amelogenin, 9 of the markers are not commonly available in other commercial kits (D7S1517, D3S1744, D2S1360, D6S474, D4S2366, D8S1132, D5S2500, D21S2055, D10S2325). Its use is highly suited for complex forensic cases, difficult paternity cases, population genetic studies among others. Here we provide the population genetic and efficiency parameters of the 12 STRs in the Investigator HDplex in mestizo Ecuadorian population. This study was preformed using blood samples in FTA cards of 200 unrelated individuals from different cities of Ecuador. The participants signed the informed consent for population based genetic studies. No significant deviation form Hardy-Weinberg Equilibrium (HW) was observed. Therefore, after the analysis preformed, the STRs under analysis constitute a powerful tool for resolving complex paternity cases in Ecuadorian population.

322 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P242 - GENETIC CHARACTERIZATION OF mtDNA CONTROL REGION FROM GYPSIES OF PAKISTAN Babar Ali1, Muhammad Farhan Khan2, Shahid Nazir2, Allah Rakha2

1 The University Of Lahore, Institute of Molecular Biology and Biotechnology, Lahore, Pakistan 2 University of Health Sciences, Department of Forensic Sciences, Lahore, Pakistan

The mtDNA sequence is one of the major attractions in forensic and genetic studies. Databases like EMPOP and NCBI lack in information of many populations, Pakistani gypsies are one of those. The control region (nucleotide position 16024–576) sequences of mitochondrial DNA were generated for 94 self-identified and unrelated gypsies from different parts of Pakistan and major haplogroups were determined. Population set showed 62 different haplotypes. Matrilineal lineages belonging to different phylogenetic origins; south Asia contributed the most and that is 53.19 % and west Asia contributed 29.7 %. The results of this population were compared to previously studied Pakistani populations and the fixation index value was found to be 0.053322 which clearly indicates that populations are different from each other. This data will serve as a reference mtDNA database for forensic casework in Pakistan.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 323 P243 - GENETIC CHARACTERIZATION OF SOUTHEAST POPULATIONS FROM BRAZIL: 32 INSERTION AND DELETION POLYMORPHISMS ANALYSIS OF THE X CHROMOSOME Flávia Alves de Jesus Silva1, Fernanda Silva Polverari1, Danilo Faustino Braganholi1, Bianca Belon Januario1, Juliana Martinez1, Isabela Brunelli Ambrosio1, Regina Maria Barretto Cicarelli1

1 Faculdade de Ciências Farmacêuticas, Ciências Biologicas, Araraquara, Brazil

The genotyping of insertion-deletion polymorphisms (InDels) has been improving importance in population genetics and human identification studies over the past few years, as well as the study of X-chromosome markers. In this study, 407 unrelated individuals, born in the states of Minas Gerais (116 men and 90 women) and 201 men from Espírito Santo, were analyzed for 32 X-InDel markers. There were no data available in the literature of X indels for these populations. The mean genetic diversity was 0.418 (±0.078) for the population of Minas Gerais and 0.438 (±0.064) for Espirito Santo. The 32 X-InDels showed for Minas Gerais and Espirito Santo respectively an accumulated power of discrimination 0.999999999994 and 0.999999999999 of in females; 0.99999994 and 0.99999998 in males; exclusion chance of in trios 0.99996 and in duos 0.9994 and 0.9998. A variant allele in the MID1361 was observed in three samples from Minas Gerais (1 female and 2 male) and a sample from Espirito Santo Female samples a genotypic distribution according to the Hardy-Weinberg equilibrium. Analysis of linkage disequilibrium revealed a significant association between some (MG: MID356-MID357, MID3690-MID3719 and MID3719-MID2089; ES: MID3719-MID2089, MID357-MID356, MID3719-MID3701, and MID3753- MID1839). The results of the analysis of the statistical parameters of forensic effectiveness suggest that the 32plex system can be used to human identification and kinship tests in the populations under study and probably in other populations as well. It is expected that these markers may also in the future assist the resolution of forensic cases in our country. All markers showed up polymorphisms for the studied populations.

324 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P244 - GENETIC DATA AND MUTATION RATES OF 23-YSTRs IN THE SÃO PAULO STATE POPULATION, BRAZIL Isabela Brunelli Ambrosio1, Nathália Andrekenas1, Danilo Braganholi1, Fernanda Silva Polverari1, Isadora Frigieri1, Januário Bianca1, Silva Flávia1, Regina Cicarelli1

1 Universidade Júlio de Mesquita Filho - UNESP, Ciências Biológicas, Araraquara, Brazil

Markers present on the Y chromosome are widely used in forensic genetics, as a complementary tool to the use of markers present in the autosomal chromosomes, used in criminal issues, mainly in sexual assault cases when there is a mixture between the samples of the aggressor and the victim, to infer kinship relations, and inferring the paternal bio-geographic ancestry. In analyzes of kinship relations, spontaneous mutations in paternal germ cells can lead to false exclusions during the analysis due to differences among the participants. Therefore, reliable estimates of Y-STRs mutation rates are necessary for the correct interpretation of Y chromosome data. We analyzed 200 cases of confirmed fatherhood in the population from São Paulo state to determine possible mutations in Y chromosome STRs, using the Powerplex® Y 23 commercial kit (Promega Corporation, USA). Twenty cases of mutations were identified in 12 of the 23-YSTRs analyzed, obtaining an average mutation rate of 4.35E-03. The markers with the highest mutation rate were the DYS576 and DYS570. In addition, we identified the amplification of three alleles in the marker DYS19, as well as the amplification of two alleles in the marker DYS389II, and the occurrence of some rare alleles and null allele. The haplotypic diversity was equal to 1. This population was classified according to its ancestral origin and resulted in 66.14 % of European origin, 28.57 % of African origin, 4.23 % of Asian origin and 1.06 % of Native American origin.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 325 P245 - GENETIC IDENTIFICATION OF SPANISH CIVIL WAR VICTIMS. THE STATE OF THE ART IN CATALONIA (NORTH‑EAST SPAIN) Sara Palomo-Díez1,2, Cláudia Gomes1,2, Ana María López-Parra1,2, Carlos Baeza-Richer1,2, Ivon Cuscó3, Caterina Raffone3, Elena García-Arumí3, Diana Vinueza Espinosa4, Cristina Santos4, Núria Montes5, Raquel Rasal6, Óscar Escala7, Juli Cuéllar Gisbert8, M. Eulàlia Subirà5, Ferran Casals6, Assumpció Malgosa4, Eduardo Tizzano3, Enric Tartera7, Gemma Domenech Casadevall8, Eduardo Arroyo-Pardo1,2

1 Laboratory of Forensic and Population Genetics- Legal Medicine- Psychiatry and Pathology Department- Medicine School- Complutense University of Madrid- Spain., Legal Medicine- Psychiatry and Pathology Department, Madrid, Spain 2 Grupo de Ciencias Forenses: Genetica y toxicología forense. Instituto de Investigación Sanitaria del Hospital Clinico San Carlos IdISSC- Madrid- Spain., Legal Medicine- Psychiatry and Pathology Department, Madrid, Spain 3 Clinical and Molecular Genetics Area- Hospital Vall Hebron- Barcelona- Spain, Clinical and Molecular Genetics Area, Barcelona, Spain 4 Laboratori d’ADN antic- Unidad de Antropología Biológica, Departamento de Biología Humana, Animal, Biología Vegetal y Ecología. Universidad Autónoma de Barcelona UAB. Bellaterra-Spain., Departamento de Biología Humana, Animal, Biología Vegetal y Ecología, Barcelona, Spain 5 GREAB. Unitat d’Antropologia Biològica. Facultat de Biociències. Universitat Autònoma de Barcelona- Spain., Unitat d’Antropologia Biològica, Barcelona, Spain 6 Servei de Genòmica- Departament de Ciències Experimentals i de la Salut- Universitat Pompeu Fabra- Barcelona- Spain, Departament de Ciències Experimentals i de la Salut, Barcelona, Spain 7 Iltirta Arqueologia SL- Corbins- Spain, Iltirta Arqueologia SL- Corbins- Spain, Corbins Lleida, Spain 8 Direcció General de Memòria Democràtica- Departament de Justícia- Generatitat de Catalunya- Barcelona- Spain, Departament de Justícia, Barcelona, Spain

At the end of 2016 and under the initiative and funding of the “Direcciò General de Memòria democratica-Departament de Justícia” (Generalitat of Catalonia), it was decided to recover and identify the remains of people disappeared in Catalonia during and after the Spanish Civil War (1936–1939). Anthropology, archaeology, history and genetics are part of the global procedure. For achieve identification by genotyping, two different genetic approaches have been: 1)the direct identification through kinship analysis of alleged living relatives, when there is an evidence (archaeological, anthropological, historical) of a possible relationship (direct identification); 2) the random crossing of a genetic profile database of victims and another database of alleged relatives in search of a possible identification (random identification). The analysis of autosomal STRs, Y chromosome STRs and, in particular cases, X-InDels and mitochondrial DNA markers have been applied in both approaches. Here, we present the state of the process in Catalonian mass graves. Nowadays a total of 102 post-mortem skeletal remains was genotyped for autosomal STRs by Global Filer Express and Y-STRs from Y-Filer Plus kit. Results were compared to actual data of living relatives in a database of 1519 genotyped for Global Filer, Y filer plus when paternally lineage was expected (248)

326 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS and mitochondrial markers when maternally lineage was expected (111). Up to now, we have identified 5 victims: 4 were identified by direct strategy (including Y-STRs and Mitochondrial markers in 2 cases) and one by random search, without any previous evidence, suggesting that both strategies are useful in this context.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 327 P246 - GENETIC INSIGHT INTO NIGERIAN POPULATION GROUPS USING AN X-CHROMOSOME DECAPLEX SYSTEM Catarina Gomes1, António Amorim1, Victoria Okolie2, Sam O Keshinro3, Anton Starke4, Carlos Vullo5, Leonor Gusmão6, Iva Gomes7

1 Institute for Research and Innovation in Health i3S- Institute of Molecular Pathology and Immunology of the University of Porto IPATIMUP- Department of Biology- Faculty of Sciences- University of Porto, Population Genetics and Evolution, Porto, Portugal 2 Lagos University Teaching Hospital LUTH, Molecular Biology Research Laboratory, Lagos, Nigeria 3 Nigeria Police Force, FCIID Annex, Lagos, Nigeria 4 University Hospital of Bonn, Institute of Medical Microbiology- Immunology and Parasitology IMMIP, Bonn, Germany 5 Argentinean Forensic Anthropology Team EAAF, DNA Forensic Laboratory, Córdoba, Argentina 6 State University of Rio de Janeiro UERJ, DNA Diagnostic Laboratory LDD, Rio de Janeiro, Brazil 7 Institute for Research and Innovation in Health i3S- Institute of Molecular Pathology and Immunology of the University of Porto IPATIMUP, Population Genetics and Evolution, Porto, Portugal

African populations have higher genetic diversity representing an interesting continent for population genetic studies. Yet some African countries remain poorly studied mainly due to the difficult access to samples. A sample set from Nigeria, a West African country on the Gulf of Guinea, was genetically characterized using 10 X chromosomal short tandem repeat polymorphisms (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789). The sample composed by 218 unrelated male individuals belong to three of the largest ethnic groups of Nigeria, namely Hausa (n=87), Yoruba (n=37) and Igbo (n=94). The presence of significant association between pairs of loci, based on an exact test of linkage disequilibrium (LD), provided no evidence for statistically significant LD (p≥0.0011) among the markers studied in the present sample. However, in small sample sizes, LD might be difficult to detect due to the low power of estimation of an exact test of LD. The genetic affinity among the three studied ethnic groups was analyzed through pairwise genetic distances (FST). No significant differences were detected (FST≤0.0013; p≥0.0773) which supports homogeneity among the three studied groups, for the X-chromosome decaplex markers. Parameters for forensic evaluation were also calculated for each X-STR further supporting the potential application of these markers in distinctive kinship scenarios where X-STR analyses may add value.

328 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P247 - GENETIC INVESTIGATION OF CHINESE SHE ETHNIC BASED ON AUTOSOMAL STRs AND X-STRs Jingyi Zhang1,2, Zihao Yang2,3, Ruiyang Tao2,4, Chengtao Li1,2, Suhua Zhang2

1 Medical School of Soochow University, Department of Forensic Science, Suzhou, China 2 Academy of Forensic Science- Ministry of Justice- China, Forensic Genetics, Shanghai, China 3 School of Basic Medical Science- Wenzhou Medical University, Department of Forensic Medicine, Wenzhou, China 4 Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Forensic Genetics, Chengdu, China

China is a multi-ethnic country embracing 56 ethnic groups, which have various origins, cultures and social customs. The She ethnic is a large minority with about 700,000 individuals. For more than 1000 years, the She ethnic migrated from the Fenghuang Mountains to Zhejiang and Fujian provinces, with a few scattered in other provinces. To obtain a better understanding of the genetic background of She, we investigated 21 autosomal STRs (A-STRs) and 16 X-STRs in 296 unrelated healthy individuals from Zhejiang She population. The STR data were generated by DNA extraction, multiple amplification, and CE genotyping. Allele frequencies and forensic efficiency parameters including heterozygosity (HET), matching probability (MP), power of discrimination (PD), polymorphism information content (PIC), power of exclusion (PE), typical paternity index (TPI) were calculated for the 21 A-STR and 16 X-STR loci, respectively. Data provides basic genetic information for forensic application, such as personal identification and parentage testing. We also compared the data with that of the previously published papers concerning other ethnic groups utilizing the same markers. Based on the respective allele frequencies of A-STRs and X-STRs, the genetic distances among different populations were calculated by Nei’s method. Ge- netic distances between She and Eastern Han are always the highest, regardless of the markers used for analysis. Although the tested STRs are located on different chromosomes with different inheritance laws, A-STRs and X-STRs provided in general congruent phylogenetic signal and same cluster among compared groups. These results demonstrated that geographic isolation and interactions play significant roles in differentiation of genetic constitution of ethnic groups.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 329 P248 - GENETIC POLYMORPHISM AND PHYLOGENETIC ANALYSIS OF 21 NON-CODIS STR LOCI IN A SHANGHAI HAN POPULATION Kuan Sun1, Zhou Zhihan1, Shao Chengchen1, Xu Hongmei1, Xie Jianhui1, Zhao Ziqin1

1 Fudan University, Department of Forensic Medicine- School of Basic Medical Sciences, Shanghai, China

Short tandem repeats (STRs) are the essential genetic markers for forensic applications, thus more and more new STR loci have been constantly discovered, studied, and applied in forensic caseworks. In the present study, we detected the 21 autosomal non-combined DNA index system (non-CODIS) STR loci in a Han population from Shanghai, China, calculated their forensic parameters and analyzed its genetic relationships with reference populations in Chinese mainland. A total of 293 alleles were observed with corresponding allele frequencies from 0.0019 to 0.5511. The cumulative power of discrimination (CPD) and the cumulative probability of exclusion (CPE) values of the 21 STR loci are 1–2.4989548480352*10-21 and 0.999999543679421, respectively. The results of inter-populations differentiation studies, phylogenetic trees and multidimensional scaling analysis indicated a closer genetic relationship between the studied population and other Han population. In conclusion, these 21 STR loci had high genetic polymorphisms in the studied Shanghai Han population, and could be used for forensic applications and studies of population genetics.

330 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P249 - GENETIC POLYMORPHISM AND POPULATION STRUCTURE OF TORGHUT MONGOLS AND COMPARISON WITH A MONGOLIAN POPULATION 3,000 KILOMETERS AWAY Riga Wu1,2, Nana Wang1,2, Dan Peng1,2, Haixia Li1,2, Hongyu Sun1,2

1 Sun Yat-sen University, Faculty of Forensic Medicine, Zhongshan School of Medicine, Guangzhou, China 2 Sun Yat-sen University, Guangdong Province Translational Forensic Medicine Engineering Technology Research Center, Guangzhou, China

Mongolians played a pivotal role in shaping the culture and genetic architecture of modern Eur- asia through the expansion of the Mongol Empire. While the historical aspects of the Mongolian Empire are well documented, research on the genetic variations among Mongolian populations is still insufficient. In this study, we examined the genetic diversity of 70 Torghut Mongols residing in China compared with 88 Jalaid Mongols residing 3,000 km away. Over 200 forensically relevant genetic markers, including autosomal STRs, X chromosomal STRs, Y chromosomal STRs, identity-informative SNPs, ancestry-informative SNPs, and phenotype-informative SNPs were genotyped. The STR genotyping results showed that 80 alleles identified in Torghut Mongols were not identified in Jalaid Mongols, while 155 alleles identified in Jalaid Mongols were not observed in Torghut Mongols. In addition, 35 alleles showed significant frequency differences (3-fold) between the two populations. Calculation of the forensic parameters demonstrated that the STRs and SNPs analyzed here could be employed in forensic applications. Interpopula- tion comparisons via principal component analysis, phylogenetic tree, and STRUCTURE analysis showed that the two Mongolian populations are closely related by their genetic background, although genetic differences were also discovered. The Y-haplogroup prediction showed that although haplogroup C2-M217 was dominant in both populations, haplogroup O2-M122 was rarely presented in Torghut Mongols. This study not only uncovered the genetic features of the two Mongolian tribes but also provide a genetic evidence that the Torghut Mongols may have developed via the gradual intermixing of nomadic groups of Mongol and Turkic origin as recorded.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 331 P250 - GENETIC POLYMORPHISM OF 125 Y-SNPs IN HAN POPULATION FROM EASTERN CHINA Yilun Zhang1,2, Tingzhi Que2, Hongran Wang3, Min Li2,4, Meng Meng3, Chengjiang Zhou1, Yingnan Bian2, Suhua Zhang2, Chengtao Li2

1 Baotou Medical College, School of Basic Medicine, Baotou, China 2 Academy of forensic science, Ministry of Justice, China, Forensic Genetics, Shanghai, China 3 Harbin Medical University, Department of Histology and Embryology, Harbin, China 4 Sichuan University, Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China

Since Y chromosome genetic markers are male-specific and follow strict paternal inheritance, they are powerful tools in forensic genetic, such as individual identification in some sexual assault cases and inferring a man’s ancestry origin. Single nucleotide polymorphism (SNP) is one of the bi-allelic genetic markers and its allele frequency is distinct between populations in different regions which proves that SNPs are associated with ancestry informative markers (AIMs). Accord- ing to the sixth National Population Census in 2010, the Han population accounts for over 90 % of the total population in China. However, studies on sub-haplogroup of the population were insufficient. In this study, a total of 118 unrelated male Han individuals in Liyang, Jiangsu Prov- ince were typed by 125 Y-SNPs including 80 sub-haplogroups of O-M175 haplogroup. The haplogroup diversity of Han population reached 0.9092 and 23 terminal haplogroups had been observed simultaneously, of which O2a1c1-F18 haplogroup (14.17 %) was the most predominant haplogroup. The results demonstrated that 1) the panel can effectively distinguish individuals; 2) most samples (85 %) from Eastern China Han in this study were genotyped into O-M175 haplogroup, known as the typical haplogroup of East Asia; 3) the number of O2-M122 sub-haplogroup is the largest in the samples.

332 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P251 - GENETIC POLYMORPHISM OF 125 Y-SNPS IN HAN POPULATION FROM SHANDONG PROVINCE, CHINA Min Li1,2, Tingzhi Que2, Yilun Zhang2,3, Lei Huang4, Jinlin Li5, Meng Meng2,6, Yingnan Bian2, Chengtao Li1,2

1 Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Forensic Genetics, Chengdu, China 2 Academy of Forensic Sciences- Ministry of Justice- China, Forensic Genetics, Shanghai, China 3 School of Basic Medicine- Baotou Medical College- Inner Mongolia Autonomous Region- China, School of Basic Medicine, Baotou, China 4 The Institute of Criminal Science and Technology, Shandong Public Security Department- China., Jinan, China 5 Institute of Criminal Science and Technology, The Public Security Bureau of Zaozhuang, Forensic Genetics, Zaozhuang, China 6 Department of Histology and Embryology, Harbin Medical University, Harbin, China

Y chromosome haplogroups have been widely used to infer paternal bio-geographical ancestry. In this study, we analyzed 125 Y-SNPs haplogroup typing panel including 36 major haplogroups from A to R and 89 O-sub-haplogroups. Total 92 male Han individuals from Shandong Dezhou were genotyped using 125 Y-SNPs markers which were divided into three multiplex reactions performed on MALDI-TOF MS platform. Haplogroup frequency and haplogroup diversity were calculated. The 92 samples were assigned to 9 major haplogroups, O haplogroup was the largest haplogroup (n=65, 71 %), followed by the relatively large haplogroups located in C-M130 (n=13, 14.1 %). These samples belonging to O haplogroup were further subdivided into 13 different terminal O-sub-haplogroups. The haplogroup diversity was estimated as to be 0.9140. Results demonstrated that the joint usage of multiplex amplification and MALDI-TOF MS could have advantages in genotyping Y-SNP markers. In addition, the panel showed high polymorphism in the Han population in Shandong, China, which has great potential in forensic application.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 333 P252 - GENETIC POLYMORPHISM OF 30 AUTOSOMAL INDEL LOCI IN CHINESE LI POPULATION FORM HAINAN Yiping Hou1, Ziwei Ye1, Jing Liu1, Hong Zhu1, Zheng Wang1

1 Sichuan University, Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Chengdu, China

A novel genetic marker, Insertion/Deletion polymorphism (InDel) shows remarkable potential for forensic DNA applications attributed to its wide distribution throughout the genome, short amplicon fragment, low mutation rate, and compatibility to common platform. Hainan Island is the southernmost and the second largest island in China, of which the Li ethnic group is regarded as the original inhabitants. In this study, 200 individual samples of Li ethnic group from Hainan were genotyped using Investigator DIPplex kit which contains 30 autosomal InDels and Amelo- genin. Allele frequency, observed heterozygosity (Ho), expected heterozygosity (He), probability of match (PM), power of discrimination (PD), power of exclusion (PE) and typical paternity index (TPI) were calculated for these loci. There was no significant deviation from the Hardy-Weinberg equilibrium. The combined power of discrimination (CPD) and the cumulative probability of exclusion (CPE) reached 0.99999999992912 and 0.9861, respectively. These results suggest that the kit was effective for personal identification in Hainan Li population.

334 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P253 - GENETIC POLYMORPHISMS OF 12 X-STRS OF INVESTIGATOR ARGUS KIT IN ECUADORIAN POPULATION Anibal Gaviria1, Anahí Boada1, Margarita Vela1, Gisella Fiallos1, Carmen Gruezo1, Cristina Rodríguez1, Juan José Builes2, Ana Karina Zambrano3

1 Laboratorio de Genética, Centros Médicos Especializados Cruz Roja Ecuatoriana, Quito, Ecuador 2 GENES Ltda, Laboratorio de Genética Forense y Huellas Digitales del DNA, Medellín, Colombia 3 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo-Universidad UTE, Quito, Ecuador

X-STR markers have shown its potential to efficiently complement the analysis in order to solve complex paternity cases. In the present study, it is reported allele and haplotype frequencies, linkage disequilibrium (LD), power of exclusion (PE), power of discrimination (PD), mean exclusion change (MEC) and polymorphism information content (PIC) of 12 X-STRs in Ecuadorian population. We examined a total of 140 unrelated individuals who had signed the informed consent to volunteered participate in genetic studies. The tested markers are in Hardy Weinberg equilibrium and can be used for match probability calculation. Moreover, Investigator Argus X-12 is applicable for Ecuadorian population in complex and routine paternity cases.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 335 P254 - GENETIC POLYMORPHISMS OF 30 INSERTION/ DELETION MARKERS IN JAPANESE, BANGLADESHI, AND INDONESIAN POPULATIONS Atsushi Nagai1, Toshimichi Yamamoto2, Masaaki Hara3

1 Gifu University, Department of Legal Medicine, Gifu, Japan 2 Nagoya University, Department of Legal Medicine and Bioethics, Nagaya, Japan 3 Saitama Medical University, Department of Forensic Medicine, Moroyama-machi, Japan

Population studies of 30 insertion/deletion (INDEL) markers were carried out with the Investi- gator DIPplex® kit (Qiagen) in 197 unrelated Japanese individuals living in the Gifu Prefecture in central Japan, 80 unrelated Bangladeshi individuals living in Dhaka (capital of Bangladesh), and 78 unrelated Indonesian individuals living in Surabaya (east region of Java Island). All the samples were used with the approval of the Ethical Committee at Gifu University Graduate School of Medicine. No significant deviations from Hardy–Weinberg equilibrium were found for 30 IN- DEL markers, with the exception of HLD77 (rs1611048; p = 0.0147) and HLD99 (rs2308163; p = 0.0205) in the Japanese samples, HLD118 (rs16438; p = 0.0273) in the Bangladeshi samples, and HLD118 (p = 0.0204) in the Indonesian samples.The highest power of discrimination (PD) was observed for HLD77 (PD = 0.6507) in the Japanese samples, HLD118 (PD = 0.6628) in the Ban- gladeshi samples, and HLD48 (rs28369942; PD = 0.6407) in the Indonesian samples. The combined PD values for 30 INDEL markers in the Japanese, Bangladeshi, and Indonesian populations were 0.999999999983, 0.999999999999, and 0.999999999937, respectively. A quantitative comparison of allele frequencies for 30 INDEL markers showed significant differences (p < 0.05) for 16 markers between the Japanese and Bangladeshi samples, 14 markers between the Japanese and Indonesian samples, and 12 markers between the Bangladeshi and Indonesian samples. The results of this study will be useful for forensic genetic investigations and population genetic studies.

336 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P255 - GENETIC PORTRAIT OF THE PUNJABI POPULATION FROM PAKISTAN USING THE PRECISION ID ANCESTRY PANEL Muhammad Adnan Shan1,2, Mie Refn1, Niels Morling1, Claus Børsting1, Vania Pereira1

1 Section of Forensic Genetics- Department of Forensic Medicine, Faculty of Health and Medical Sciences- University of Copenhagen- Frederik V’s vej 11- 2100, Copenhagen, Denmark 2 Centre for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan

Ancestry-informative markers (AIMs) are genetic markers that give information about the biogeographic ancestry of individuals. There is a growing desire to apply ancestry inference in forensic casework when biological material is recovered from a crime scene, but no suspect or match in a relevant crime DNA database is found. Punjab is the second largest and most populous of the four provinces of Pakistan. It has a population of more than 90 million corresponding to 46 % of the Pakistani population. Punjab is located in the northwestern part of the Indian plate at the Indus River system. Various ethnic groups settled in this region and formed the Indus Valley Civilization in the bronze age 3,300 to 1,300 BCE. The Punjabi population is the largest ethnic group in Pakistan. It consists of a heterogeneous population group with various tribes, clans, and communities. The native language of the province is “Punjabi”. Exploration of AIMs in Pakistan is interesting due to the distinct subpopulations with multidi- rectional ancestry from neighboring states and native groups. As part of an ongoing project on the characterization of Pakistani subpopulations, DNA from 88 unrelated individuals from Pun- jab was investigated with the Precision ID Ancestry panel. The Arlequin v.3.5 software was used to calculate various population genetic parameters, and principal component and STRUCTURE analyses were used to compare the results of the Punjabi individuals with those of individuals from neighboring populations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 337 P256 - GENETIC VARIATION OF 23 STR LOCI IN A NORTHEAST COLOMBIAN POPULATION (DEPARTMENT OF SANTANDER) Adriana Castillo1, Adriana Pico1, Adriana Gil1, Leonor Gusmão2, Clara Vargas1

1 Universidad Industrial de Santander, Laboratorio de Genetica, Bucaramanga, Colombia 2 Universidade do Estado do Rio de Janeiro, Laboratorio de diagnostico por DNA, Rio de Janeiro, Brazil

Colombia is a country divided into 32 departments, one of which is Santander located on the Northeast region of the country. There are many different groups in the country, coming from different continents, thus being considered a multi-ethnic country. In the present study, we have collected 123 samples from unrelated individuals from Santander. These samples were genotyped for the 23 STRs included in the PCR Amplification multiplex Verifiler Express kit (Thermo Fisher Scientific), namely D3S1358, vWA, D16S539, CSF1PO, TPOX, Y indel, Amelogenin, D8S1179, D21S11, D18S51, Penta E, D2S441, D19S433, TH0, FGA, D22S1045, D5S818, D13S317, D7S820, D6S1043, D10S1248, D1S1656, D12S391, D2S1338 and Penta D. Amplified products were separated and detected using an ABI 3500 sequencer, and alleles were assigned with the Gene- mapper IDX v. 1.5. Statistical analyses included Hardy-Weinberg tests and calculation of parameters of forensic relevance: observed and expected heterozygosities, mean exclusion chance, discrimination power and possible associations between loci. All markers are in Hardy-Weinberg equilibrium (exact p-values ≥ 0.0716). The locus Penta E presented the highest power of discrimination (PD=0.9884), and D2S1338 and D6S1043 loci showed the highest power of exclusion (PE=0.8004). The accumulate power of exclusion for the full set of 23 STR marker-set was high (99.9996 %), as well as the match probability, which was 1 in 8.77E+29. The results obtained showed that all studied markers have high values of diversity in the population of Santader, and the Verifiler Express kit seems to be a useful tool for forensic identification in this region.

338 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P257 - GENETIC VARIATION OF 25 Y-CHROMOSOMAL AND 15 AUTOSOMAL STR LOCI IN THE HAN CHINESE POPULATION OF LIAONING PROVINCE, NORTHEAST CHINA Jun Yao1, Bao-jie Wang1, Jia-xin Xing1, Jin-feng Xuan1, Xi Xia1

1 China Medical University, School of Forensic Medicine, Shenyang, China

In the present study, we investigated the genetic characteristics of 25 Y-chromosomal and 15 autosomal short tandem repeat (STR) loci in 305 unrelated Han Chinese male individuals from Liaoning Province using AmpFISTR® Yfiler® Plus and IdentifilerTM PCR amplification kits. Population comparison was performed between Liaoning Han population and different ethnic groups to better understand the genetic background of the Liaoning Han population. For Y-STR loci, the overall haplotype diversity was 0.9997 and the discrimination capacity was 0.9607. Gene diversity values ranged from 0.4525 (DYS391) to 0.9617 (DYS385). Rst and two multi-di- mensional scaling plots showed that minor differences were observed when the Liaoning Han population was compared to the Jilin Han Chinese, Beijing Han Chinese, Liaoning Manchu, Liaoning Mongolian, Liaoning Xibe, Shandong Han Chinese, Jiangsu Han Chinese, Anhui Han Chinese, Guizhou Han Chinese and Liaoning Hui populations; by contrast, major differences were observed when the Shanxi Han Chinese, Yunnan Bai, Jiangxi Han Chinese, Guangdong Han Chinese, Liaoning Korean, Hunan Tujia, Guangxi Zhuang, Gansu Tibetan, Xishuangbanna Dai, South Korean, Japanese and Hunan Miao populations. For autosomal STR loci, DP ranged from 0.9621 (D2S1338) to 0.8177 (TPOX), with PE distributing from 0.7521 (D18S51) to 0.2988 (TH01). A population comparison was performed and no statistically significant differences were detected at any STR loci between Liaoning Han, China Dong, and Shaanxi Han populations. The results showed that the 25 Y-STR and 15 autosomal STR loci in the Liaoning Han population were valuable for forensic applications and human genetics, and Liaoning Han was an independent endogenous ethnicity with a unique subpopulation structure.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 339 P258 - GENETIC VARIATION OF HIGH-ALTITUDE ECUADORIAN POPULATION USING AUTOSOMAL STR MARKERS Ana Karina Zambrano1, Aníbal Gaviria2, Carmen Gruezo2, Cristina Rodríguez2, Isaac Armendáriz‑Castillo1, Jennyfer M. García-Cárdenas1, Patricia Guevara-Ramírez1, Santiago Guerrero1, Paola E. Leone1, Andrés López-Cortés1, Andy Pérez-Villa1, Verónica Yumiceba1, Gisella Fiallos2, Margarita Vela2, César Paz-y-Miño1

1 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo- Universidad UTE, Quito, Ecuador 2 Laboratorio de Genética, Centros Médicos Especializados Cruz Roja Ecuatoriana, Quito, Ecuador

Ecuador is a country located in South America, bordering the Pacific Ocean on the west, Colom- bia in the north and Peru in the east and south. Furthermore, according to the last census (2010) projection, the Ecuadorian population is 17 267 936, and it is divided in 24 provinces distributed in four main regions: coast (8 523 453 individuals), highland or the Andes (7 733 725 individuals), the Amazonia (937 406 individuals) and island regions or Galapagos (32 320 individuals). The altitude of Ecuadorian cities differ from 4 m.a.s.l. to 3264 m.a.s.l. For this high diversity, the aim of the present study is to investigate the forensic statistical parameters and genetic diversity of high-altitude Ecuadorian population of 15 autosomal STRs commonly used. There have been published data of STRs genetic statistical parameters for Ecuadorian population, but there are no reports of Ecuadorian population at different altitudes. This study provides a dataset of statistical forensic parameters of high-altitude Ecuadorian population. Besides, it also reported a population comparisons between high-altitude and low-altitude Ecuadorian.

340 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P259 - GENOMIC PORTRAIT OF POPULATION OF UTTAR PRADESH, INDIA, DRAWN WITH AUTOSOMAL STRs AND Y-STRs Ankit Srivastava1, Vijay Kumar Yadav1, Kriti Nigam1, Shivani Dixit2, Ramkishan Kumawat3, Divya Shrivastava4, Pankaj Shrivastava2

1 Bundelkhand University- Jhansi - 284128- UP- India, Dr. APJ Abdul Kalam Institute of Forensic Science & Criminology, Jhansi, India 2 State Forensic Science Laboratory- Sagar-470001- Madhya Pradesh- India, DNA Fingerprinting Unit, Sagar, India 3 State Forensic Science Laboratory- Jaipur-302016- Rajasthan- India, DNA Division, Jaipur, India 4 Jaipur National University- Jaipur-302017- Rajasthan- INDIA, School of Life Sciences, Jaipur, India

Genetic portrait of population of Uttar Pradesh is drawn by analysing autosomal and Y- STRs in 100 unrelated male individuals to establish parameters of forensic interest. Allele frequencies are reported for autosomal STRs. The examined autosomal STRs revealed high combined power of exclusion (CPE) and combined power of discrimination (CPD). The haplotype diversity, discrimination capacity and matching probability for Y-STRs loci is also reported.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 341 P260 - GEOLOCATION OF THE BRAZILIAN NATIONAL DNA DATABASE MATCHES AS A TOOL FOR IMPROVING PUBLIC SAFETY AND THE PROMOTION OF JUSTICE Ronaldo Carneiro da Silva Junior1, Aline Costa Minervino1, Luciano Lamper Martinez2, Daniel Russo2, Daniel Araújo Miranda2

1 Brazilian Federal Police, Technical Scientific Directory, Brasilia, Brazil 2 Brazilian Federal Police, National Institute of Criminalistics, Brasilia, Brazil

Created in 2013, the Brazilian National DNA Database (BNPG) has been growing yearly. In 2018 there was a 68 % increase in the number of genetic profiles that joined BNPG. Along with this growth, there was also a 150 % increase in the number of matches that occurred at the national level. While that increase is an excellent sign of increasing database effectiveness, it brings new challenges. One of them is how to monitor and organize the matches so that they become an effective tool for the promotion of justice and public safety. The solution to this was found within our institution, through the integration of matches data from BNPG with Inteligeo - a geolocation system developed by Brazilian Federal Police. Launched in 2010, Inteligeo is a modern and powerful geographical information system, which assists Brazilian forensic experts in the production of technical reports and integrated information management. The teams of BNPG and Inteligeo started a joint work to implement the project. First, all forensic DNA laboratories and their respective matches were geolocated. Correlations between laboratories and matches distribution maps were created. A protocol was also developed to made it mandatory to register the geographical coordinate of the sites that originated the samples related to matches. The possibilities of this data integration are many, considering that Inteligeo allows integration with external data sources, supports complex geospatial searches and allows the generation of dynamic maps that can be used to instantly assess the general status of the database and view the correlations of individual matches as they occur. At this point, the team works on the expansion of the geolocated data in order to extend the potential of this tool.

342 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P261 - HAPLOTYPE DATA FOR THE 12 RM Y-STR LOCI IN A SYRIAN POPULATION Mustafa Ay1, Ayse Serin1, Necmi Cekin1

1 University of Cukurova, Forensic Medicine, Adana, Turkey

Researches with RM Y-STRs have shown that these loci provide substantially higher haplotype diversity and haplotype discrimination capacity in worldwide populations when compared with the YSTRs commonly used in genetic forensics. The aim of this study was to develop an allelic frequency database for the Syrian population living in Turkey in order to obtain population data of 12RM Y-STRs. A total of 80 unrelated males from the Syrian population living in Turkey were typed with 12 RM Y-STRs loci: DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS526a/b, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. A high RM Y-STR haplotype diversity was found 1.00 in these samples. The highest GD was observed for the locus DYF399S1 (0.986), followed by loci DYF404S1 (0.92) and DYF387S1 (0.87). Based on the results of this study, the RM YSTR loci showed remarkable haplotype resolution power in the Syrian population, high genetic diversity and, therefore, demonstrating their usefulness in forensic identiﬁcation cases.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 343 P262 - HUMAN TRAFFICKING – MULTINATIONAL CHALLENGE FOR FORENSIC SCIENCE Magdalena Buś1, Tim Schellberg2, Bruce Budowle3

1 Centre for Human Identification, University of North Texas Health Science Centre- 3500 Camp Bowie Blvd. Fort Worth- TX 76107- USA, Fort Worth, USA 2 Gordon Thomas Honeywell Governmental Affairs, 1201 Pacific Ave- Suite 2100- Tacoma- Washington 98402- USA, Washington, USA 3 Center for Human Identification, University of North Texas Health Science Center- 3500 Camp Bowie Blvd. Fort Worth- TX 76107- USA, Fort Worth, USA

Human trafficking encompasses diverse forms of human exploitation such as sex trafficking, labor exploitation, marriage, begging, child soldiers, and illegal organ transplantation. Millions of people are subjected to current-day slavery worldwide. The consequences of human trafficking affect all countries, are devastating for victims and society and impact social, economic, and global health costs. A project led by the UNTCHI is underway to assist Guatemala, El Salvador, Honduras, Costa Rica, and Panama to identify the missing and combat human trafficking on a multinational level by developing model legislation and policy, and an effective DNA forensic science capability and database systems. The focus is on at-risk populations and the most vulnerable of society. This program engages the public and government in establishing legislative models for DNA identification of missing persons that protects the privacy of individuals such as family members who donate reference samples, maintains security of the databases, and properly limits database search parameters. Implemented regulations for managing and operating a DNA database must follow international standards of privacy and human rights. Concomitantly, laboratory personnel must be trained, procedures validated, and work performed under quality assurance practices. The laboratory and the database must be sustainable for long term use. The work described herein can be an example for transferring technology and database practices, establishing policy and legislation, engaging the public, and developing strategies for sustainability to other vulnerable countries. The approach can help combat human trafficking within the countries and promote ultimately multi-national exchange of DNA data to identify the missing.

344 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P263 - IMPROVED DNA-DATABASE SEARCH WITH MIXED PROFILES USING SMARTRANK (1) Martin Eckert1

1 Bundeskriminalamt, KT31, Wiesbaden, Germany

At present, the German DNA-Database holds more than 1.2 Million profiles from convicted offenders, suspects and crime scene stains. Due to the database structure there is no option for registration of mixed profiles. Currently, only high-quality two person mixtures can be searched. In order to search the database with more complex mixtures that represent the majority of recent typing results we conducted a pilot-study on the feasibility of implementation of Smar- tRank. This biostatistic tool has been shown to very well establish putative contributor listings by using likelihood ratio calculations. This study yielded a number of promising results, even when a rather large database was used. The testing experiences will be presented together with challenges in overall workflow organization. The findings of this study provide a solid ground for other laboratories considering improvement of mixtures searches. (1) Benschop CCG, et al.: Validation of SmartRank: A likelihood ratio software for searching national DNA databases with complex DNA profiles. Forensic Sci Int Genet. 2017 Jul;29:145–153.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 345 P264 - INCREASING CONVICTED OFFENDER GENETIC PROFILES IN THE BRAZILIAN NATIONAL DNA DATABASE – LEGISLATION, PROJECTS AND PERSPECTIVES Ronaldo Carneiro da Silva Junior1, Aline Costa Minervino1

1 Brazilian Federal Police, Technical Scientific Directory, Brasilia, Brazil

According to Brazilian law, genetic profiles of individuals are obligatorily included in DNA Da- tabases in cases of conviction for heinous/intentional violent crimes or by means of judicial authorization. Although expected since 2012, the obligation to identify the genetic profile of convicted offenders under Law 12,654 has not been effectively fulfilled. In December 2017, Brazilian National DNA Database (BNPG) had a little more than 2,000 convicted offenders genetic profiles, which represented a very small percentage of the prison population. In addition, the strategic planning of the Ministry of Justice and Public Security for the quin- quennium 2015–2019 predicted as a goal increasing the number of collections to 50 % of those convicted offenders under law. In early 2018 it was carried out the “DNA Laboratories Survey”, which counted on 100 % participation of the Brazilian Forensic DNA laboratories. The survey showed that there are in Brazilian prisons about 137,600 convicted offenders that need to be genetically identified. In order to subsidize the formulation of protocols, a Working Group was established. Based on its guidance, the Ministry has invested in a project based on some pillars: 1. Survey of needs and potentialities of each laboratory; 2. Definition of protocols and training of personnel; 3. Acquisition of inputs and equipments; 4. Political management to promote Law 12,654/2012 compliance; 5. Budget transfers to states linked to the project adhesion; 6. Monitoring of collections and insertions by BNPG administrators. As a result of the work carried out in 2018, at the beginning of 2019 a large increase in the number of convicted offenders collections (>18,000) and profiles (>8,000) was observed. The goal is 65,000 convicted offender profiles in late 2019.

346 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P265 - INFERRING THE GENETIC STRUCTURE OF NORTHWESTERN ARGENTINA BY UNIPARENTAL SNP TYPING Maria Josefina Castagnola1, Hortensia Cano2, Maria Laura Hulaniuk3, Julieta Trinks3, Daniel Corach4, Mariela Caputo4

1 Universidad de Buenos Aires- Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Genética, Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG., Buenos Aires, Argentina 2 Poder Judicial de Santa Cruz, Laboratorio Regional de Investigación Forense, Rio Gallegos, Argentina 3 Instituto Universitario. Hospital Italiano, Basic Science and Experimental Medicine Institute ICBME, Buenos Aires, Argentina 4 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Genética, Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG. CONICET, Buenos Aires, Argentina

The genetic background of South American populations is the result of three major genetic admixture events involving Native Americans, Europeans and West Sub-Saharan Africans. To investigate the maternal and paternal lineages, we analyzed mitochondrial DNA (mtDNA): A2, B2, C, D1; and Y-chromosome haplogroups (hg): R1b1b2, Q1a3a, G2a, I, J2, E1b1b, based on SNP typing by real time PCR and high resolution melting analysis. Individuals from Northwestern Argentina (Jujuy n=87) and Catamarca (n=100) were studied and compared with Buenos Aires´ inhabitants (n=107) and recently arrived immigrants from Bolivia (n=100), Paraguay (n=54) and Peru (n=52). Amerindian mtDNA hg were the most frequent in all regions (89,8 % to 98,1 %), except for Buenos Aires (43 %), being B2 the most prevalent (42,6 % to 58 % versus 8,4 % in Buenos Aires). Regarding Y-hg, a greater contribution of the non-native lineage was found in all populations (61,5 % to 99 %), except for Bolivia (32,7 %). Moreover, the most frequent hg observed were R1b1b2 (50 % Paraguay, 46,6 % Buenos Aires, 30,2 % Northwestern Argentina) and Q1a3a (67,3 % Bolivia, 38,5 % Peru). In Northwestern Argentina, there were no statistically significant difference between Catamarca and Jujuy provinces’ native and non-native groups for both uniparental markers, although a major proportion of mt-DNA hg D was detected in Catamarca (20 % versus 1,14 % Jujuy) (p<0,0001). A high percentage of native mt-DNA/non-native Y-DNA was represented (53,8 % to 63,5 %), excepting except for Buenos Aires (41,7 %) and Bolivia (26,9 %). The conclusions are consistent with historical information underscoring the complex genetic ancestry of melting pot countries and provides additional tools for forensic investigation; molecular epidemiological and anthropological studies.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 347 P266 - INTRODUCING A FORENSIC VERSION OF PHYLOTREE FOR IMPROVED HAPLOGROUPING Arne Dür1, Nicole Huber2, Walther Parson2,3, The dna.bases Consortium

1 Institute of Mathematics, University of Innsbruck, Innsbruck, Austria 2 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 3 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

Mitochondrial DNA haplogrouping is an important tool that is not restricted to forensic mitochondrial genome (mitogenome) analysis. In many population genetic studies, haplogroup determination is a decisive prerequisite for further investigations and research questions.In general, haplogroup determination is based on the nomenclature provided by Phylotree. The latest version (build 17) uses 24,275 full mitogenomes, resulting in a tree with 5,435 different haplogroup defining motifs. However, an increasing number of sequences do not truly fit into the tree and numerous existing variants have not been used to characterize the respective clades.Based on 24,928 reviewed full mitogenomes we refined the original haplogroup motifs of Phylotree 17 without changing the haplogroup nomenclature. In total, we modified ~750 motifs through realignment or addition of frequently observed variants. Moreover, we added ~800 potential subclades with at least two representing genomes.Subsequent evaluation was carried out on an internal data set containing more than 100,000 mitogenomes as well as on previously published data. The results obtained with the refined version of Phylotree 17 showed more precise haplogroup assignment for both the full mitogenome and its control region.In summary, with the refined version of Phylotree 17 we achieved a 16 % increase of haplogroup defining motifs for full mitogenomes and a 30 % increase for the mtDNA control region that is particularly of forensic interest. This work received support from the European Union grant agreement number 779485-STEFA - ISFP-2016-AG-IBA-ENFSI.

348 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P267 - LISBON POPULATION mtDNA ANALYSIS: STUDY OF A GENETIC MARKER WITH POPULATION, EVOLUTION AND FORENSIC INTEREST Heloísa Afonso Costa1,2, António Amorim1,3, Cláudia Vieira Da Silva1, Maria João Porto1, Eugénia Cunha1,4, Manuel Carlos Lemos2, Francisco Corte Real1,5

1 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Serviço de Genética e Biologia Forenses, Lisboa, Portugal 2 Universidade da Beira Interior, Faculdade de Ciências da Saúde, Covilhã, Portugal 3 Universidade de Lisboa, Faculdade de Ciências, Lisboa, Portugal 4 Universidade de Coimbra, Faculdade de Ciências e Tecnologia Forenses, Coimbra, Portugal 5 Universidade de Coimbra, Faculdade de Medicina, Coimbra, Portugal

Mitochondrial DNA (mtDNA) is remarkable in population genetic studies to clarify the history and demographic past of human populations. For forensic purposes mtDNA analysis is particularly useful when there are degraded or low level DNA samples. Nonetheless, only being in possession of the genetic reference data of the populations it is possible to attribute statistical weight to an evidence in forensic casework. The National Institute of Legal Medicine and Forensic Sciences in Portugal carried out a study with the aim of portraying the genetic diversity of immigrants living in Lisbon. However, the nonimmigrant population of Lisbon, the reference population, had not yet been studied. The present study intends to determine the mtDNA variability of the Lisbon metropolitan native population. A total of 90 unrelated individuals were sampled. The entire control region of the mtDNA was amplified with primers L15971 and H639 and sequenced with BigDye® Terminator v.3.1 Cycle Sequence and primers L15971, L16555, H016 and H639. Sequences were detected in a 3130 Ge- netic Analyzer. Results were analyzed and compared with revised Cambridge Reference Se- quence in SeqScape v.3. The frequency of haplotypes was calculated by direct counting using EMPOP database. Among the 90 samples, 87 unique mtDNA haplotypes were identified. It was verified that 32 of the haplotypes had no coincidence and 14 of the haplotype had only one coincidence on EM- POP database. This low frequency of the majority of the haplotypes emphasizes the importance of studying this population. The most frequent haplotype, enclose haplogroup R0 with a frequency of 2.0211e-2. The haplotypes studied belong predominantly to west Eurasian mtDNA. Were also identified haplotypes that belong to Haplogroups L0, L1, L2, L3 and D.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 349 P268 - LOW DISCRIMINATION POWER OF THE YFILER™ PLUS PCR AMPLIFICATION KIT IN AFRICAN POPULATIONS. DO WE NEED MORE RM Y-STRs? Chiara Della Rocca1, Eugenio Alladio2, Filippo Barni2, Francesco Cannone2, Eugenia D’Atanasio1, Beniamino Trombetta1, Andrea Berti2, Fulvio Cruciani1

1 Sapienza Università di Roma, Dipartimento di Biologia e Biotecnologie “C. Darwin”, Rome, Italy 2 Reparto Carabinieri Investigazioni Scientifiche di Roma, Sezione di Biologia- Viale Tor di Quinto 119- 00191, Rome, Italy

Recently, thirteen rapidly mutating Y-STRs (RM Y-STRs), characterized by a mutation rate higher than 10-2/STRs/generation, have been proven to be extremely useful in distinguishing among close male relatives. Six of these RM-YSTRs have been included in the YFilerTMPlus PCR Ampli- fication Kit (ThermoFisher Scientific), which shows the highest discriminatory power among Y-STRs commercial multiplex currently available.In this study, we used YFilerTMPlus to analyze 1437 males from eastern, northern and sub-Saharan Africa (462, 477 and 498 subjects from 20, 11 and 14 populations, respectively). 242 out of 1437 subjects were found to share 102 Y-STRs haplotypes, resulting in a low discrimination capacity (DC = 0.90) as compared to other continental regions. Y-STRs haplotype sharing was limited to subjects coming from the same country and ethnic group, and belonging to the same binary Y-SNPs haplogroup, suggesting possible familial relationships along the male lineage. In order to evaluate the presence of hidden familial relationships in our sample set, all the 242 subjects sharing a Y-STRs haplotype were further genotyped for 16 autosomal STRs using the Amp- FℓSTR® NGM SElect™ PCR Amplification Kit (ThermoFisher Scientific). A blind search for kinship using the Familias software (v. 3.2.5) revealed the presence of a low number of close relatives in our sample set. These findings show that close relatedness explains only a small proportion of the observed Y-STRs haplotype sharing, suggesting that a higher number of RM Y-STRs should be included in commercial kits to improve the discrimination power of male-specific markers in regions characterized by high levels of endogamy.

350 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P269 - MASSIVELY PARALLEL SEQUENCING OF FORENSIC MARKERS IN THE PEOPLE OF THE BRITISH ISLES Tunde Huszar1, Walter Bodmer2, Katarzyna Hutnik2, Jon Wetton1, Mark A. Jobling1

1 University of Leicester, Department of Genetics and Genome Biology, Leicester, United Kingdom 2 University of Oxford, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom

The People of the British Isles (PoBI) project captured the autosomal genetic variation of rural areas of the UK, based on SNP chip analysis of 2039 individuals. The PoBI set aimed to represent local ancestry, relatively unaffected by most recent migrations, by recruiting individuals with ancestors known to inhabit the same local areas for at least two generations. From this collection we selected 362 male samples covering 44 sub-regions to analyse the sequence level variation in autosomal and Y-STR markers, as well as the mitochondrial DNA (mtDNA) control region, using the prototype PowerSeq™ Auto/Mito/Y System (Promega) massively parallel sequencing (MPS) assay. Here we provide a population-based description of the 22 autosomal STRs and 23 Y-STRs analysed and the mtDNA haplotypes observed across the sampled regions. We highlight the pattern of the observed specific sequence variants and summarise and contrast the distribution characteristics of maternal and paternal landscapes. Our dataset provides the first overview of commonly used forensic markers at the DNA sequence level in the British Isles, and the potential usefulness of MPS-based approaches in assessing population sub-structure.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 351 P270 - MATERNAL GENETIC ANCESTRY OF CROATS: POPULATION GENETICS AND FORENSIC PERSPECTIVE Lucija Barbarić1, Bettina Zimmermann2, Korana Lipovac1, Viktorija Sukser1, Sara Rožić1, Marina Korolija1, Walther Parson2

1 Forensic Science Center “Ivan Vučetić”, Biology, Zagreb, Croatia 2 Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria, National DNA Database Laboratory, Innsbruck, Austria

Present-day Croatia, a country of Southeastern Europe, is characterized by homogenous ethnic composition and complex demographic history. In order to evaluate Croatian maternal lineages and to establish the national forensic mitochondrial DNA (mtDNA) reference database, complete control region (CR) sequences were obtained from 200 randomly selected individuals. In total, 156 different haplotypes were found, which represent a haplotype diversity of 0.9984 and a random match probability of 0.66 %. Moreover, Croatian mitochondrial pool is defined by Westeurasian lineages, except for one sample classified into East Asian lineage A. The most common haplogroups were H (25.5 %), U (22 %), followed by T (10 %), K, J, R (7.5 % each) and HV (6 %), which accounted for almost 90 % of the total variability. Results confirmed the assumption that mtDNA profiles of Croats resemble those of their geographical neighbors. Besides population genetic studies, the dataset will be used in mtDNA applications in forensic and missing persons casework.

352 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P271 - MATERNAL GENETIC CHARACTERIZATION OF A COLOMBIAN ANDEAN POPULATION Adriana Castillo1, Lara Deccache2, Elizeu Fagundes Carvalho2, Filipa Simão2, Leonor Gusmão2

1 Universidad Industrial de Santander, Basic sciences, Bucaramanga, Colombia 2 Universidade do Estado do Rio de Janeiro, Laboratorio de Diagnostico por DNA, Rio de Janeiro, Brazil

The composition of most American populations involves the admixture between Native Ameri- cans, who inhabited the region before the European colonization, Europeans and Africans, who were brought to America as slave labor. The genetic composition of Colombian populations results from the admixture between these three population groups and tends to have a high genetic diversity. In the light of this, the aim of this study was to obtain an overview about maternal lineages composition, and diversity, from the Andean populations of Colombia. Then, a sample of 100 unrelated individuals from this region were selected for sequencing the control region of mtDNA, and to calculate haplotype diversity and haplogroup composition. Preliminary results from 38 samples were used to calculate FST genetic distances between our sample and published data from South American admixed populations from Colombia, Peru, Argentina, Paraguay and Brazil. No significant differences were observed between our sample and the Colombian population deposited on 1000genome Project. On the other hand, high FST genetic distances were obtained in the pairwise comparison between our sample and Peru, Argentina, Paraguay and Brazil. A haplotype diversity of 0.9900 ± 0.0088 was observed with 4 haplotypes being shared between 2 individuals and one haplotype shared between 3 individuals. 95 % of the haplotypes belong to halogroups found both in central and south America (A2, B2d, C1, D1 and D4). The remaining 5 % (2 samples) belong to the African haplgroups L3.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 353 P272 - MITOCHONDRIAL CONTROL REGION VARIATION IN THE SOUTHERN AFRICAN POPULATION SAMPLE SET Michelle Livesey1, Peter Gustav Ristow2, Maria Eugenia D’Amato1

1 The University of the Western Cape, Biotechnology, Bellville, South Africa 2 Roche Sequencing Solutions, Research and Development Scientist, Cape Town, South Africa

South Africa`s demographic complexity has been historically shaped by interethnic admixture between the native KhoiSan inhabitants and an event known as the Bantu expansion started 3000 - 5000 YBP and reached South Africa about 1000 years ago. This followed by the arrival of the European settlers in the 16th century who brought in slaves from their Asian colonies, mainly from Malaysia and the east- and western Africa. A high-quality dataset of the complete mitochondrial DNA (mtDNA) control region (nucleotide position 16024–576) sequences were generated with Sanger and Next generation sequencing method, with the intention to characterize the maternal genetic ancestry of 248 individuals residing in the Western Cape, Northern Cape and KwaZulu-Natal province of South Africa. Results show a total of 97 haplotypes characterized by 119 polymorphic sites, of which 68 haplotypes were unique. The gene diversity and the probability of two random individuals showing identical mtDNA haplotypes were 0.952 and 0.051, respectively. The haplogroups were assigned utilizing HaploGrep2 online platform. Our data revealed higher rates for the Native African ancestry of 87,90 %, followed by European and Asian, both making up a portion of 6.05 %. The structure of this population, reinforce the importance of an increased database to capture the high diversity that is generally associated with admixed populations and heterogeneity in geographic distribution of lineages. The matrilineal lineages of South Africa will be made available through EMPOP at the end of this study.

354 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P273 - MITOCHONDRIAL DNA SEQUENCING FOR FORENSIC HUMAN IDENTIFICATION ON A SAMPLE OF SAUDI POPULATION Dinah Aloraer1, William Goodwin1

1 University of central lancashire, Forensic and applied sciences, Preston, United Kingdom

Mitochondrial DNA (mtDNA) has been used in forensic genetics for over 25 years, but its use in many countries is limited. This is largely due to the cost involved along with the low information content in comparison with autosomal STRs. However, with the wider use of massive parallel sequencing (MPS) the analysis of mtDNA is being re-evaluated by laboratories that have not adopted the methodology to date. This technology has the potential to increase the information content of mtDNA profiling and speed of analysis. The creation of regional reference databases is recommended for all forensic loci, but even more important for lineage markers that show more pronounced geographical variation than STR markers. While there is already a large amount of mtDNA sequence data produced from different global and regional populations, limited mtDNA data exists for the Kingdom of Saudi Arabia, with most previous studies focussing on either medical aspects or population groups, and so do not necessarily reflect the population at large. This study aims to analyse in the region of 500 samples collected from Saudi nationals. Sequenc- ing of the hypervariable regions will be followed by whole genome sequencing, using MPS. Ap- proximately 500 samples have been collected from King Fahad Medical city in Riyadh and DNA extracted. The D-loop has been amplified and sequenced using Sanger Sequencing; the next stage of the project involves using a MPS platform to carry out whole genome sequencing. This will allow the frequency of different haplotypes and haplogroups to be evaluated in the Saudi population and act as a reference database in casework.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 355 P274 - MITOCHONDRIAL DNA STUDY IN THE SHUAR ETHNIC GROUP FROM ECUADOR Paola E. Leone1, Diana Maldonado Oyervide1,2, Odalis Astudillo González1,2, Andy Pérez-Villa1, Verónica Yumiceba1, Isaac Armendáriz-Castillo1, Jennyfer M. García-Cárdenas1, Santiago Guerrero1, Patricia Guevara-Ramírez1, Andrés López-Cortés1, Ana Karina Zambrano1, César Paz-y-Miño1

1 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador 2 Unidad de Posgrado Investigación y Desarrollo, Universidad de Guayaquil, Guayaquil, Ecuador

Ecuador is a multiethnic country, composed of 21 indigenous groups, among these the Shuar, who are settled in around 668 communities in different provinces of the Ecuadorian amazon region (Provinces: Napo, Pastaza, Morona Santiago, Zamora Chinchipe, Sucumbíos and Orel- lana), the coast region (Provinces: Esmeraldas and Guayas), as well as in the Peruvian amazon state. Population census showed that 80 thousand individuals belong to this ethnic group, from which 22 thousand live in the Sucumbios Province. In order to know the ethnic history of the Ec- uadorian Shuar population, the mitochondrial hypervariable region was studied from the Shuar communities, Kumbatza and Yukateis, which are located in the Morona Santiago province, Ec- uador. 55 individuals, nonrelated by surnames and family tree, were analyzed, by following all the ethic needs and consents. PCR amplification and sequencing of the HV1 and HV2 regions were performed; Sequence alignment, by using the Cambridge reference sequence, identified haplotypes for each individual and, thus, the haplogroups. Result analysis showed that the Shuar population exhibited the haplogroup B which belongs to the Native American population. This study is the first in characterizing the Ecuadorian Shuar ethnic group, it describes the presence of one of the founder haplogroups from the Native Americans and demonstrates that the Shuar are a conserved population with no mixing with the European and African diaspora populations from Ecuador.

356 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P275 - MITOCHONDRIAL GENETIC PROFILE OF THE YORUBA POPULATION FROM NIGERIA Beatriz Martinez1, Masinda Nguidi2, Maria Laura Catelli3, Carlos Vullo3, Victoria Okolie4, Sam Keshinroe5, Elizeu Fagundes Carvalho2, Leonor Gusmão2, Filipa Simão2

1 University of Cartagena, Institute for Immunological Research, Cartagena, Colombia 2 DNA Diagnostic Laboratory LDD, State University of Rio de Janeiro UERJ, Rio de Janeiro, Brazil 3 DNA Forensic Laboratory, Argentinean Forensic Anthropology Team EAAF, Cordoba, Argentina 4 Molecular Biology Research Laboratory, Lagos University Teaching Hospital LUTH, Lagos, Nigeria 5 FCIID Annex, Nigeria Police Force, Lagos, Nigeria

Nigeria is located in the Gulf of Guinea and is considered as the “giant of Africa”, being the seventh most populated country in the world. Nigeria has a great genetic and cultural diversity with at least 500 ethno/linguistic groups. Yoruba is the second major group (and the most spoken language), and its populations are concentrated in the southern region of the country. Aiming at the construction of a forensic mitochondrial DNA database, the total mtDNA control region was analyzed from 71 Yoruba samples. A total of 65 unique haplotypes were found and the haplotype diversity was 0.9976 ± 0.0028. FST genetic distances were calculated, including North, East, Central-West and Southwest African populations. The sequences showed no significant differences with the Yoruba samples from the 1000 Genome database (FST = - 0, 00148, p =

0.5598) and with the geographically closest population of Ghana (FST= 0.0124, p = 0.01386). In this study, we are reporting that Yoruba population from Nigeria have a mitochondrial genetic profile similar to other populations belonging to West Africa region, probably due to continuous gene flow and ethnic affinities with these neighboring populations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 357 P276 - MOLECULAR VARIANTS ASSOCIATED WITH FLAVOR PERCEPTIONS AND ANCESTRAL PROPORTIONS OF ECUADORIAN POPULATION César Paz-y-Miño1, Jennyfer M. García-Cárdenas1, María Emilia Dávalos1,2, Ana Carolina Cárdenas Figueroa1,2, Isaac Armendáriz-Castillo1, Santiago Guerrero1, Patricia Guevara- Ramírez1, Andrés López-Cortés1, Andy Pérez-Villa1, Verónica Yumiceba1, Ana Karina Zambrano1, Paola E. Leone1

1 Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador 2 Facultad de Ingeniería y Ciencias Agropecuarias, Universidad de Las Américas, Quito, Ecuador

The perception of taste is determined by several factors, including genetics which at the same time is related with the diet and diseases in different populations. We aimed to identify the frequency of 6 single nucleotide polymorphisms (rs307355, rs35744813, rs713598, rs1726866, rs10246939 and rs11091046) involved in the perception of sweet, bitter and salty flavor in Ecua- dorian population. It was found that the presence of the T allele of rs307355 and rs35744813 is associated with decreased ability of subjects to carry out specific discrimination of sweet- ness, this is particularly interesting given the correlation found (p=0.0022) between sucrose perception and family history of cancer and diabetes. Furthermore, rs713598, rs1726866 and rs10246939 do not influence the ability to perceive the bitter taste, however we found difference between women and men and their ability to detect bitterness in the lowest concentration (0.001 % of sodium benzoate, p=0.040), which is also related to alcohol consumption (p=0.02). Concerning the perception of the salty taste, rs11091046 does influence the capacity of detecting it more easily. It was interesting to establish that genotypic frequencies in Ecuador were very similar to Europeans and Asians, although the majority of participants were predominantly Native Americans. This could be explain due to the genetic result of miscegenation and ethnic influences. This theoretical knowledge of the genetic influence on taste perception can contribute to the understanding of eating habits and their impact on human health.

358 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P277 - MUTATION RATE ANALYSIS OF 24 AUTOSOMAL STR’s MEASURED ALONG A 10 YEARS PERIOD IN ARGENTINA Andrea Sala1, Mariela Caputo1, Daniel Corach1

1 Universidad de Buenos Aires- Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Génetica. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG - CONICET., Buenos Aires, Argentina

We investigate the mutation rates of autosomal STRs in paternity tests including trios or duos, in our laboratory, along a period of ten years. Were analyzed 5986 parent-child meiosis including 24 autosomal STRs. These STRs corresponds to the expanded CODIS core of 20 STRs plus loci Penta D, Penta E, SE33 and D6S1043. We detected mutations in 21/24 loci in 132 meiotic segregations events: 93 were paternal mutations, 19 maternal and 20 undetermined. The proportion of paternal vs maternal mutations was 5.7:1 and the age of parents range from 20 to 55 years old. Simple STRs accounted for 61/132 of overall mutations, 58/132 to compound and 13/132 to complex STRs. The highest mutation rates were observed at CSF1PO, D12S391, D16S539, D18S51, D21S11, D3S1358, D8S1179 and FGA accumulating 62 % of the total (70 % of them paternally transmitted). Mutation rate ranged from 0 to 0.0018. Lowest mutation rate was observed in THO-1 and TPOX (average mutation rate 1.6E-04 and 3.3E-04, respectively). All mutations can be addressed to gain/ loss of one repeat unit, single step mutations. In addition, there were observed three mutations compatible with null alleles and three tri-allelic patterns. No mutations were detected at loci D2S441, D22S1045 and D10S1248. This work is the first attempt to generate a local database of autosomal STRs mutation for the population of Argentina to be used in forensic genetic analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 359 P278 - MUTATION RATES INVESTIGATION OF 27 Y-STRs IN 101 FATHER-SON PAIRS Jiashuo Zhang1,2, Longfei Miao3, Lu Qiao3, Jingyi Zhang1,2, Ruiyang Tao2,4, Hui Hu3, Xuewei Xu3, Shaodong Shan3, Suhua Zhang2, Chengtao Li1,2

1 Medical School of Soochow University, Forensic Genetics, Suzhou, China 2 Academy of Forensic Sciences, Ministry of Justice, China, Forensic Genetics, Shanghai, China 3 Material Evidence Authentications and Research Center of Dezhou Public Security Bureau- China, Forensic Genetics, Dezhou, China 4 Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Forensic Genetics, Chengdu, China

Rapidly mutation Y chromosome STRs (RM Y-STRs) have recently been applied in forensic community and commercial kits were accordingly developed. The mutation rate investigation of these Y-STRs is quite necessary for forensic application, such as kinship analysis and familial searching. In this study, we try to explore the mutation rates of Y-STRs from Y-filer® Plus Kit in 101 father-son pairs from the Shandong Han population of China. Y-filer® Plus Kit is a six-dye multiplex PCR kit which can simultaneously amplify 27 Y-STR targets. Among the 27 Y-STRs, seven RM Y-STRs were included. The 101 pairs of father and son parental relationship were confirmed by autosomal STRs analysis. With Y-STRs analysis, 101 meiosis from fathers to sons were observed, in which 16 mutations were found at DYF387S1a/b, DYS391, DYS438, DYS449, DYS518, DYS533, DYS570 and DYS576. The average mutation rate was 0.0059. The longer alleles are more likely to mutate, compared to the shorter alleles at each locus. Mutations at DYS449 and DYF387S1 were both found in one pair. Except the two double-step mutations that occurred at DYS570 and DYF387S1, all remaining mutations were single step; in other words, 87.5 % mutations were single step. A significant difference between the number of gains (N=3) and losses (N=13) of repeats were also observed. These mutation rates were also compared with the cumulative mutation rates available at the YHRD website. All confidence intervals were overlapped, and no significant difference was found.

360 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P279 - NGS-BASED MICROHAPLOTYPE GENOTYPING: 124‑PLEX ASSAY, ALLELE-CALLING SOFTWARE AND PROPOSED ALLELE NOMENCLATURE Le Wang1, Chi Zhang2

1 Institute of Forensic Science- Ministry of Public Security- China, Division of Forensic Science Innovation and Development, Beijing, China 2 Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, Ministry of Public Security, Beijing, China

Microhaplotype, defined as the combination of two or more SNPs within a segment of DNA up to 300 base pairs, is an emerging type of molecular markers in the field of forensic genetics. Microhaplotype possesses the advantages of both STR and SNP markers and has important application prospects in forensics including mixed DNA genotyping, ancestry inference and complicated kinship analysis. Next generation sequencing, capable of genotyping microhaplotype markers and directly yielding the phase, has been expected to become the major method for microhaplotype detection. Here we developed an in-house multiplex assay of 124 microhaop- lotypes based on next generation sequencing. The Illumina Truseq strategy was employed for library preparation, and the 124 microhaoplotype markers were simultaneously sequenced on a Miseq machine. Raw data quality, intro- and inter-locus balances were evaluated for this assay, and 256 Chinese Han individuals were sequenced. 513 alleles were observed for the 124 mi- crohaoplotypes. Ae values for 28 loci were above 3. Forensic parameters, including MP, PD, PE, were calculated, and linkage relationship among different loci were analyzed. In addition, we developed a software named MHTyper for microhaoplotype allele-calling, sequencing depths statistics and plotting. The perl-based MHTyper software used FastQ or Bam files as input files, and was open for analysis parameter adjustment and panel design. The accuracy of this software had been verified manually with the Integrative Genomics Viewer. We also propose for discussion a numeral nomenclature of microhaplotype alleles for efficient forensic applications, which has been integrated into the MHTyper software.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 361 P280 - NOVEL APPROACHES TO POPULATION GENETIC ANALYSES OF X-CHROMOSOMAL DATA – EXEMPLIFIED USING A NORWEGIAN POPULATION SAMPLE Daniel Kling1, Andreas Tillmar2

1 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway 2 National Board of Forensic Medicine and Forensic Toxicology, Department of Forensic Genetics, Linköping, Sweden

This study provides an insight into untraditional approaches to study X-chromosomal population data. In particular we focus on how to measure linkage disequilibrium for multiallelic markers as well examples of how to evaluate the potential of these markers. We use simulations to study some common kinship cases where the X chromosome is an important tool. We provide an interpretation of the simulations to assess the potential of the markers. We use a new data set comprising 631 unrelated Norwegian males typed for 12 X STRs (Argus X12, Qiagen). We compare the data set to existing populations (where data is available) and use different methods to evaluate whether this data set can be merged with adjacent populations thus creating a larger and potentially better database. The results indicate that the haplotypes gathered from the Norwegian population sample can be merged with other proximal population thus yielding a considerably larger pool of haplotypes. Given the limited amount of information available in relation to the true haplotype frequency distribution, we believe such measures are important to obtain more robust likelihood ratios not relying on small and insufficient background information.

362 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P281 - ON THE STATE GENOME REGISTRATION IN THE RUSSIAN FEDERATION Irina Perepechina1

1 Lomonosov Moscow State University, Department of Criminalistics of the Faculty of Law, Moscow, Russian Federation

The objective to create the DNA database in the Russian Federation had been set in 1994 in the Federal Programme of the Russian Federation for the Strengthening the Fight against Crim- inality for 1994–1995. The Forensic Science Center of the Ministry of the Interior of the Russian Federation was put in charge of the implementation of the project. The scientific concept and fundamental practical provisions on the creating the federal DNA database in Russia have been developed by Perepechina et al. (1995–1997). The Forensic Science Center launched an integrating activity regarding domestic forensic DNA analysis labs, thus initiating the standardization of the methods used in different labs. In the mid 1990s an experimental DNA database as a ten- tative model of the future state DNA database was formed on the basis of the Forensic Science Centre and some regional DNA labs of the Ministry of the Interior. In 1995 the Forensic Science Center joined the DNA Working Group of the European Network of Forensic Science Institutions (ENFSI). In 2008 the federal law «On the state genome registration in the Russian Federation» was adopted which became the legal base of the federal database of genome information (FDBGI) created in 2009. Genome information for FDBGI is supplied by DNA labs functioning in the Forensic Science Center and 60 regional expert forensic units of the Ministry of the Interior, together with forensic DNA labs functioning in the Investigative Committee of the Russian Federation and in the forensic medical service of the Ministry of Health (Storozhenko, 2017). The registration of genome information is carried out using home combined federal search system of genetic identification «Xenon-2» on the basis of the examination of 20 polymorphic loci plus Amelogenin.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 363 P282 - OPTIMIZATION OF THE GENOGEOGRAPHER’S REFERENCE POPULATION FOR GREENLAND Helle Smidt Mogensen1, Vania Pereira1, Torben Tvedebrink2, Poul Svante Eriksen2, Niels Morling1

1 University of Copenhagen, Forensic Medicine- Section of Forensic Genetics, Copenhagen, Denmark 2 Aalborg University, Department of Mathematical Sciences, Aalborg, Denmark

Ancestry informative markers (AIMS) are genetic markers that give information about the ancestry of an individual. The current Greenlandic population has a large variation in the degree of genetic admixtures between the original migrants from Siberia and Alaska, and the Europe- an settlers. Such a genetic landscape of a reference population can result in uncertainty when addressing the origin of a genetic profile of an individual. For genetically homogenous populations, a reference sample size of 75 individuals is adequate for reliable allele frequency estimates. However, when analyzing 793 AIM profiles of individuals from Siberia, South/Central Asia, and East Asia, the GenoGeographer did not reject 71 of the AIM profiles (~9 %), and instead, attributed the most likely population of origin of these individuals to Greenland. This indicates that in the absence of a more adequate reference population, a small percentage of individuals with Asian and European admixture are not rejected as being Greenlanders. In order to optimize the GenoGeographer’s ancestry inference, we typed additional samples from Greenland and added the profiles to the Greenlandic reference population, now consisting of 236 Greenlanders from East and West Greenland. We reanalyzed the 793 samples with the GenoGeographer. After the inclusion of the new profiles, 59 AIM profiles (~7 %) could still not be rejected as coming from Greenland. This result shows how intricate ancestry inference in admixed individuals/populations can be, and highlights the importance of population references when calculating such estimates.

364 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P283 - POLYMORPHISM ANALYSES FOR CHINESE HAN POPULATION USING FORENSEQTM DNA SIGNATURE PREP KIT Qingqing Du1, Lihong Fu1, Chaolong Lu1, Bin Cong1, Shujin Li1

1 College of Forensic Medicine, Hebei Medical University, Department of Forensic Genetics, Shijiazhuang- Hebei province, China

In contrast to capillary electrophoresis (CE), massively parallel sequencing (MPS) has the advantage of testing much more markers including short tandem repeat (STRs) or single nucleotide polymorphism (SNPs) simultaneously, alongside obtaining more genetic information such as sequence variation of repeat motif and flanking region exist in the loci. Here, 50 Chinese Han individuals lived in Hebei province were genotyped using ForenSeqTM DNA Signature Prep Kit including 27 autosomal STRs, 24 Y-STRs, 7 X-STRs and 94 identity-informative SNPs (iiSNPs). The sequencing data were analyzed by STRait Razor v2s. The results show that there are 219 length-based alleles for 27 autosomal STRs, which increases to 303 and 312 alleles under considering sequence variation of motif and flanking regions, respectively. Seven autosomal STRs and 14 iiSNPs are found to have flanking region variation. Continuing with analysis for X-STR and Y-STR, 51 and 120 length-based alleles are observed, while taking into account isoalleles, 10 and 26 alleles are added in, respectively. However, there are only 3 flanking region variations in sex chromosome STRs. Combining of the 27 autosomal STRs, random match probability (RMP) of length-based, motif sequence-based and flanking region-based are 9.25×10-31, 1.61×10-33 and 1.94×10-34, power of exclusion for trios (PEtrio) are 0.999999999961, 0.99999999999833, and 0.99999999999942, respectively. While combined with 94 iiSNPs, length-based and sequence-based RMP decline to 1.04×10-65 and 4.02×10-71, respectively.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 365 P284 - POPULATION DATA FOR 29 BIALLELIC MARKERS ON Y CHROMOSOME FROM THREE GROUPS OF CHINESE HAN INDIVIDUALS Li Li1, Linan Zhang2, Yutong Song2, Ruxin Zhu1

1 Academy of Forensic Science, Forensic Biology, Shanghai, China 2 East China University of Political Science and Law, Forensic Biology, Shanghai, China

Objective: 29 biallelic markers on Y chromosome were typed in order to compare three groups of Chinese individuals within Han population. Method: 29 Y chromosomal biallelic markers that are polymorphic in Han males in Eastern China were selected. A total of 343 unrelated male samples from the Eastern Chinese Han ethnic group were genotyped by a combination of MALDI-TOF MS (a previously developed typing system) and microsequencing-based SNaPshot assay. For SNaPshot typing, genomic DNA was extracted by Chelec-100 method from blood samples and then analyzed for four multiplex PCR anplifi- cation system followed by single base extension reactions. Allelic frequencies, gene diversity (GD) for each locus and haplotype diversity for the panel were then determined. Comparison between different populations was performed using Arlequin software. Results: All the 29 loci showed high polymorphism GD≥0.2). A total of 64 haplotypes were detected. The haplotype diversity reached 0.9010. After performing allele frequencies comparison among the Han population in three regions, 17 of the 29 loci showed significant p-value. Com- parison showed significant differences between: Eastern Han and Northwestern Han at two loci (rs2032665, rs2267801), Eastern Han and Southern Han at four markers (rs16980601, rs16980610, rs2032665, rs52812045), Southern Han and Northwestern Han at 17 loci (rs11096432, rs11096433, rs16980363, rs16980426, rs16980601, rs16980610, rs16980641, rs17174528, rs17269928, rs2032665, rs2032674, rs2267801, rs4589047, rs52812045, rs9306845, rs9306848, rs9786707). Conclusions: Some differences are shown with the loci used in the present study, which reveals that the biallelic markers panel on Y chromosome maybe useful for differentiation purposes within Chinese Han populations.

366 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P285 - POPULATION DATA OF 12 X-STRs IN TURKEY Ayse Serin1, Esen Kalaoglu1, Mustafa Ay1, Ayca Ulubay1, Behnan Alper1

1 University of Cukurova, Forensic Medicine, Adana, Turkey

X-chromosomal short tandem repeats (X-STRs) have been proven to be useful in case of deficiency paternity testing in which the mother is available for typing, the possible X alleles of the putative father can be determined and the paternal profile can be reconstructed. In this study, we examined 144 (91 female, 53 male) samples taken from unrelated healthy people living in Turkey with the Investigator Argus X-12 kit (Qiagen, Germany) which detects 12 X-STR markers. The highest GD value of the 12 X-STR loci was DXS10135 (0.98), while the lowest was 0.87 for DXS7433. Evidence of Linkage Disequilibrium (LD) was detected in this population data within each linkage group. The most discriminative linkage group (LG) was LG1 (GD=0.989). Combined power of discrimination was calculated as over 0.9999999999 in females and in males by including LD information. Single duplication was also seen in 3 samples in the loci DXS1079 and DXS10103. Allele and haplotype frequencies as well as different forensic statistical parameters of the 12 X-STR loci tested indicated that they are highly informative in different forensic applications in Turkey population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 367 P286 - POPULATION DATA OF D6S1043, PENTA D AND PENTA E LOCI IN SOUTH OF ITALY (CALABRIA) Anna Barbaro1, Cormaci Patrizia1, Angelo La Marca1

1 Studio Indagini Mediche E Forensi SIMEF, Forensic Genetics, Reggio Calabria, Italy

In the present paper, we studied population data of D6S1043, Penta D and Penta E loci in 230 unrelated healthy natives of Calabria (South Italy). Calabria region still preserves a mixture of different cultures because during centuries it has been greatly influenced by Greek, Roman, Arabic civilizations and then in the modern age by Spanish and French dominations. Forensic statistical parameters were calculated by STRAF software, Hardy-Weinberg equilibrium and other population data were estimated using Arlequin software v.3.5.2.2. Results showed that all 3 STRs were highly polymorphic: Penta E was the most informative locus (based on Het, PIC, PD and TPI), while D6S1043 was the less informative. Comparison with previously published population data showed that Penta E allele frequencies were very similar to Sicilian ones. This study confirms that D6S1043, Penta D and Penta E loci are useful for forensic purposes especially if analyzed in combination between them or in association with other loci available in commercial STR multiplexes.

368 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P287 - POPULATION GENETIC DATA AND FORENSIC STATISTICAL PARAMETERS OF 23 STR AUTOSOMAL MARKERS IN ARGENTINA Nicolás Furman1, Malena Canteros1, Luciana Rabitti1, Franco Marsico1, Marcela Cepeda1, Mariana Herrera Piñero1, Walter R. Bozzo1

1 Banco Nacional de Datos Geneticos, Ministerio de Educación, Cultura, Ciencia y Tecnología, Buenos Aires, Argentina

Object: we determined allelic frequencies and forensic statistical parameters for 23 STR autosomal loci in 949 samples of Argentinean unrelated donors. Methodology: autosomal STR profiles from peripheral blood samples were obtained by using PowerPlex Fusion 6C System and GlobalFiler kits. Fragment length analysis was done by capillary electrophoresis using an ABI 3500 Genetic Analyzer and GeneMapper ID-X 1.2 software. The allelic frequencies, Hardy–Weinberg equilibrium, observed and expected heterozygosity were determined by using Arlequin V3.5.2.2 software. Forensically relevant statistical parameters of population genetics were evaluated by calculating Power of Discrimination (PD), Polymorphic Information Content, Typical Paternity Index (TPI) and Power of Exclusion (PE) with the Familias v3.2.2 software. Results: none except one autosomal STR (Penta D) departed from Hardy–Weinberg equilibrium, even after applying Bonferroni correction (0.05/23 = 0.002). The SE33 locus shows the highest value for discrimination and exclusion power. The lowest values were detected in TPOX and D22S1045 respectively. Conclusions: these results allow us to use our population allele frequencies for individual identification and paternity testing. Further studies must be done regarding Penta D system in order of knowing whether the HW disequilibrium could be explained by population substructure or the presence of null alleles.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 369 P288 - POPULATION GENETICS DATA OF 23 AUTOSOMAL STR LOCI FOR THREE POPULATIONS IN UNITED ARAB EMIRATES Mohammed Naji1,2, Ruksar Damji2, Rashed Alghafri1,2, Synan Abu-Qamar2

1 Dubai Police, Forensic Department, Dubai, United Arab Emirates 2 United Arab Emirates University, Biology, Alain, United Arab Emirates

Forensic parameters were estimated for three populations resident United Arab Emirates including UAE Arabs, Pakistani and Indian, based on the population data of 23 autosomal short tandem repeats (STRs) loci. UAE Arabs population is an important population due to high con- sanguineous marriage. So, it is important to estimate the alleles distribution within this population. In addition, it is crucial to study the majority individuals of the population including Indian and Pakistanis. A total of 1272 blood samples were collected on FTA® cards, comprising 571 UAE Arabs, 352 Indians and 349 Pakistanis. All of these samples were amplified directly using Verifiler® Express PCR Amplification Kit which includes, D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, D10S1248, D1S1656, D12S391, D2S1338, D6S1043, Penta D and Penta E loci. PCR products were run on the ABI 3500 Genetic Analyzer and analyzed using GeneMapper ID-X software. Arlequin v3.5 and Forstat softwares were used to determine the forensic parameters and population structure using AMOVA. A total of 804 alleles were observed with the corresponding allelic frequencies ranging between 0.000876 and 0.49387. Gene diversity ranged from 0.67406 (TPOX) to 0.9226 (Penta E). The most variable autosomal STR loci observed was Penta E (observed heterozygosity: 0.91244, match probability: 0.0147). Results showed the 23 STR loci had a relatively high genetic variation, suggesting suitability for forensic personal identification and kinship analysis in the relevant populations. The significance of this work is to build an allelic frequency database for one of the most powerful commercially available STR amplification kits using current forensic workflow.

370 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P289 - POPULATION GENETICS OF 30 INSERTION–DELETION POLYMORPHISMS IN POLISH POPULATIONS USING INVESTIGATOR® DIPPLEX KIT (QIAGEN) Eliza Michalak1, Monica Abreu-Glowacka1, Magdalena Konarzewska2, Witold Pepiński3, Małgorzata Skawrońska3, Monika Wójcik3, Ireneusz Sołtyszewski4, Czesław Żaba1

1 Poznan University of Medical Sciences, Department of Forensic Medicine, Poznan, Poland 2 Medical University of Warsaw, Department of Forensic Medicine, Warsaw, Poland 3 Medical University of Bialystok, Department of Forensic Medicine, Bialystok, Poland 4 University of Warmia and Mazury in Olsztyn, Department of Criminalistics and Forensic Medicine, Olsztyn, Poland

The objective of this study was to estimate the diversity of 30 InDel markers in three Polish subpopulation samples and to evaluate their usefulness in forensic genetics practice. Buccal swabs were collected from 389 unrelated volunteers dwelling in three Polish geo-historical regions: Greater Poland (n=168), Mazovia (n=113) and Podlasie (n=108), along with information on the birthplace and ethnicity of the donor. The extracted DNA was amplified with use of In- vestigator® DIPplex® Kit (Qiagen) that contains 30 autosomal DIP markers. The InDels frequency distributions showed no deviations from HWE (Bonferroni corrected). Among Poles, the mean heterozygosity value is 0.4081, and the combined matching probability value is 6.05x10(-12). In- Del genotyping constitutes a robust, sensitive and very informative identification system highly applicable to routine forensic casework. The highly polymorphic systems exhibit high informativeness and are a potential extension to STR loci, particularly in kinship analysis and deficiency cases.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 371 P290 - POPULATION GENOMICS OF THE MITOCHONDRIAL GENOME SEGMENTS AND THE PREDICTION OF NEUTRAL AND SELECTIVE TRENDS FOR IDENTIFICATION AND ASSOCIATION STUDIES Gian Carlo Iannacone De La Flor1

1 Legal Medicine Institute, Operation Management, Lima, Peru

Whole mitochondrial genome (mtDNA) was analysed by segments between populations with the aim to finding segments with neutral or selective trend, applied in the field of forensic identification and association studies(population adaptation). For this purpose, an algorithm was developed comprising three indexes: Segment weight index, Segment weight distribution index and Combined segment index. These indexes were analyzed with 3250 mtDNA (47 Populations -1000 genomes and HGDP databases). A total of 576 segments were found in the mtDNA with a minimum limit between segments ≥6 nucleotides without variation. Then, at the global population level, we found four types of segments that correspond to a trend of: Selective–S (49 %), Slightly Selective–SS (10 %), Slightly Neutral–SN (25 %) and Neutral–N (15 %). The SN and N are frequent in respiratory genes and some tRNA. Likewise, the SN and N are the largest in size (mean 104pb vs 54pb in S and SS). The multivariate analysis between macropopulations and mtDNA segments showed a differential structure for Combined Segment Index S(<-1.5), SS(- 1.5 to 0), SN(0 to 1.5) and N(>1.5) by macropopulations which correspond to their contribution in the global population genetic density (nMDS stress=0.07 and ANOSIM R=42). For example, the Latinamerican macropopulation is the second with greater SN and N diversity, where the Pe- ruvian population has the highest coding mtDNA haplotype (98.7 %) and also for the SN and N. However, Colombia population have lower coding mtDNA haplotype diversity (95.8 %) but it is similar at the level of diversity of SN and N to Peruvian population. Then, it show independence population histories within the coding mtDNA haplotype and it is relevant tool for forensic identification (SN and N) and association (S and SS).

372 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P291 - POPULATION SUB-STRUCTURE WITHIN THE BRITISH ISLES DETECTED WITH Y-STR AND Y-SNP MARKERS Jon Wetton1, Gurdeep Lall1, Jodie Lampert1, Walter Bodmer2, Katarzyna Hutnik2, Gianpiero Cavalleri3, Richard Jones4, Joanna Story4, Mark A. Jobling1

1 University of Leicester, Department of Genetics and Genome Biology, Leicester, United Kingdom 2 John Radcliffe Hospital, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom 3 Royal College of Surgeons in Ireland, Molecular and Cellular Therapeutics, Dublin, Ireland 4 University of Leicester, Department of History, Leicester, United Kingdom

We have undertaken a survey of the diversity of paternal lineages within the British Isles using the Promega PowerPlex Y23 Y-STR multiplex. Analysis of 873 men of known regional origin reveals marked underlying sub-structure between some of the constituent nations (England, Wales and the Republic of Ireland), islands (Orkney) and even English counties, with declining haplotypic diversity with distance from mainland Europe. This mirrors patterns detected in high resolution autosomal SNP surveys which are believed to reflect the influence of invasion and migration from the near continent upon existing tribal territories during the first millennium CE, and are here supported by comparison with population samples from Friesland (N=91) and Nor- mandy (N=90) as proxies for the origins of Anglo-Saxon and Norman invaders. Within Y-SNP haplogroups, either known or predicted using software validated against a larger European dataset, we detect clusters of similar haplotypes comprising 73 % of men from the British Isles which correlate with the three most common Y-SNP haplogroups - I1, R1a and R1b. Dating of the clusters suggests shared origins for each cluster of around 3–5KYA, whilst the R1b-M222 sub-haplogroup which is particularly common within Ireland radiated approximately 1.5KYA. Regional differences in haplogroup frequencies and their recent and distinct origins contributes to significant variation in the average similarity of haplotypes within and between populations. Whilst samples drawn from major urban centres are representative of the population as a whole and reflect the high discrimination power of full profiles, the estimation of match probabilities for partial profiles is likely to be affected by the unique demographic histories of peripheral regions.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 373 P292 - PRELIMINARY ASSESSMENT OF THE DNASEQEX 29 Y-STR PANEL DESIGNED FOR THE MISEQ FGX® Xiaoqin Qian1, Jonna Muller2, Steffi Bredemeyer3, Martin Fabiani4, Marie-Louise Kampmann1, Rick de Leeuw2, Sascha Willuweit3, Meghan Didier4, Kathryn Stephens4, Walther Parson5, Niels Morling1, Yiping Hou6, Lutz Roewer3, Cydne Holt4, Claus Børsting1, Peter de Knijff2

1 Section of Forensic Genetics, Department of Forensic Medicine- Faculty of Health and Medical Sciences- University of Copenhagen, Copenhagen, Denmark 2 Department of Human Genetics, Leiden University Medical Centre, Nijmegen, Netherlands 3 Institute of Legal Medicine and Forensic Sciences, Charité-Universitätsmedizin Berlin- corporate Member of Freie Universität Berlin- Humboldt-Universität zu Berlin- and Berlin Institute of Health, Berlin, Germany 4 Verogen Inc., Verogen Inc., San Diego, USA 5 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 6 Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Sichuan University, Chengdu, China

The use of capillary electrophoresis (CE) based Y-STR profiling is well established in forensic genetics as a method for kinship analysis, mixture analysis and population genetics. However, fragment analysis by CE has constrained multiplexing capacity and is unable to identify isometric alleles, which reduces the true power of discrimination of Y-STRs. Massively parallel sequencing offers an alternative detection method that overcomes the limitations of CE. We present a collaborative study to evaluate a Verogen prototype multiplex of 29 Y-STRs proposed by the DNASEQEX project. The panel works with the existing ForenSeq DNA Signature Prep Kit reagents and workflow. It amplifies the 17 Y-STRs contained in the ForenSeq® DNA Sig- nature Prep Kit (DYF387S1a/b, DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS437, Y GATA H4, DYS438, DYS439, DYS448, DYS481, DYS570, DYS576, DYS612 and DYS635) and a further 12 Y-STR loci (DYS710, DYS644, DYS627, DYS626, DYS547, DYS526, DYS518, DYS458, DYS456, DYS449, DYS393, DYF404S1) selected by DNASEQEX. Samples were run in Copenhagen and Leiden and included a sensitivity test, controlled male/female mixtures, a set of 8 deep rooting pedigrees and a series of globally dispersed human males. Analyses were performed using a custom pipeline (Charité), FDSTools (Leiden), and STRinNGS (Copenhagen). The prototype was developed for characterization purposes only and does not represent a fully optimized system. However, the results from this study will be used to assess the potential utility of the additional Y-STRs proposed by DNASEQEX and whether they could be used independently and/or incorporated into future commercially available multiplexes. Variations in Y-STRs and their flanking regions will be described and future directions discussed.

374 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P293 - RAPIDLY MUTATING Y-STRs FOR QATARI POPULATION Eida Almohammed1, Rashed Alghafri2, Fatma Sorror1, Sibte Hadi3

1 Ministry of Interior Qatar, Qatar Forensic Laboratory, Doha, Qatar 2 Dubai Police G.H.Q., Department of Forensic Sciences and Criminology, Dubai, United Arab Emirates 3 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom

Differentiating male lineages using non-recombining Y-chromosomal genetic markers is highly informative for tracing human migration and for forensic studies. Recently, it has been shown that the level of male lineage resolution can be enhanced by analysing Rapidly Mutating (RM) Y-STRs. The aim of this study was to develop an allelic frequency database for Qatari population to evaluate the resolution power of 13 RM Y-STRs. The overall haplotype diversity (HD) was 100 %. It was found that the markers which contributed the most toward high HD were DYF399S1 and DYF403S1a/b. Together with their value for paternal male relative differentiation, these RM Y-STRs will be a valuable asset for forensic casework. AMOVA test was performed between Qatari population in comparison to Gulf countries, Middle East, and several worldwide population data sets. FST values were also calculated. Geography was found to account considerably for the pattern of population substructuring. The RM Y-STR markers showed remarkable haplotype resolution power in the Qatari population, high gene diversity and sufficient robustness for a diverse range of applications.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 375 P294 - RARE GENETIC STRUCTURE OBSERVED IN POPULATION GENETIC ANALYSIS OF THE GLOBALFILER STR LOCI IN THE GHANAIAN POPULATION Pet-Paul Wepeba1,2, Arati Iyengar3, William Goodwin1

1 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom 2 Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, School of Medical Sciences, Kumasi, Ghana 3 Albany- State University of New York, Biological Sciences, New York, USA

While many publications have reported population STR data for many regions of the world, population level studies are limited for countries in sub-Saharan Africa. This study profiled 1,047 volunteers using the GlobalFiler™ Kit from the four main ethnic groups in Ghana: the Akan (282), Mole-Dagomba (253), Ewe (250) and Ga-Dangbe (262), representing about 98.4 % ethnic coverage of the Ghanaian population. The polymorphic nature of the African population was demonstrated during the profiling of the reference data set, with the occurrence of some rare variant alleles. At the TPOX locus, three triplet alleles were found; the allelic pattern 6- 8–10 occurring twice and 9–10–11 once. At the D5S818 and D3S1358 loci; 11–13–14 and 15–16–17 tri-allelic patterns were seen respectively. Although the TPOX tri-allelic pattern has been reported in other populations including Afri- can, the D5S818; 11–13–14 has never been reported in an African population. It has however been reported once with a frequency of 1 in 69,600 in a convicted offender case in the USA. The D3S1358; 15–16–17 pattern has not been reported in an African population. In conclusion, the occurrence of rare alleles is illustrative of the genetic diversity within the Gha- naian population. This was further illustrated by the presence of three individuals that were heterozygous at all 21 STR loci.

376 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P295 - RELATED ANALYSIS OF SURNAME AND Y CHROMOSOME HAPLOTYPE IN THE JAPANESE POPULATION Eriko Ochiai1, Kiyoshi Minaguchi1, Masato Nakatome2, Motoki Osawa1

1 Tokai University School of Medicine, Department of Forensic Medicine, Isehara, Japan 2 Faculty of Medicine Tottori University, Division of Legal Medicine, Yonago, Japan

The non-recombinant Y chromosome, except the short telomere side, reveal the male lineage. The haplotype of polymorphisms like SNP and STR has served in forensic personal investigation and anthropology. By contrast, surname of father’s family has been given to babies historically in Japan as similar as other oversea societies. Surname is one of the major group markers in any society. In this study, we report the relation of Japanese surname and Y chromosomal polymorphisms. Buccal cells and blood were obtained from 329 Japanese male volunteers who had major 19 surnames with informed consent. Y haplotype was determined based on 15 STR markers of the AmpFLSTR Y-filer PCR Amplification Kit (Thermo Fisher Scientific) and 34 SNPs of the HID-Ion AmpliSeq Identity Panel (Thermo Fisher Scientific) or Sanger sequencing. Phyloge- netic analysis combined STR and SNP was performed in a provided algorithm. This project has been approved by the ethical committee of Tokai University School of Medicine. As a result of AMOVA analysis of Y-STR haplotypes and calculation of pairwise Rst values, significant differences were found among the groups of some surnames, indicating various degrees of genetic divergence among the surnames. In Y-SNP haplogroup, the compositional ratio of haplogroup D and O in each surname was different depending on the surname. In STR stepwise phylogeny, the haplogroup D and O1b sample groups in some surnames formed a subpopulation in which differences in STR haplotypes are within 5 step mutations. Furthermore, those subpopulations were the same subgroup in the Y haplogroup. The number of examined samples was limited, but we showed the close genetic relationships in some surnames. Y chromosome haplotyping will serve male lineage analysis in forensic investigation.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 377 P296 - REVISITING THE MATRILINEAL LINEAGES AND HYPOXIC ADAPTATION OF HIGHLAND TIBETANS Mengge Wang1, Guanglin He1, Yiping Hou1, Zheng Wang1

1 Sichuan University, Institute of Forensic Medicine, Chengdu, China

The mitochondrial DNA (mtDNA) plays a vital role in forensic, anthropological, biogeographical and genealogical studies. In the present study, we sequenced 59 mitochondrial genomes of Tibetan individuals settling in Muli Tibetan Autonomous County of Sichuan Province using the Precision ID mtDNA Whole Genome Panel and Ion S5 XL system. Meanwhile, 192 published complete mitogenomes from five Tibetan populations wereincluded for further analysis. All 251 investigated Tibetan participants were assigned to 98 unique subclades pertained to the macrohaplogroups M and N, and 17 subhaplogroups were considered as major haplogroups of Tibetans since these subhaplogroups accounted for considerably high frequencies in randomly selected Tibetans. It was noteworthy that M9a1a1c1b1a was the predominant sub- haplogroup in the Tibetans collectively. Furthermore, the nonsynonymous substitutions and synonymous substitutions ratios (N/S) of Tibetans, Tibetan highlanders (Monpas, Lhobas, Dengs and Sherpas), non-Tibetan highlanders and general populations were estimated to evaluate the potential selective constraints. The N/S ratio in the Tibetan groups (0.503) is higher than that in Tibetan highlanders (0.465), non-Tibetan highlanders (0.430) and general populations (0.415). The distributions of N/S ratio in 13 protein-coding genes revealed that significant differences were existed in COX2, ATP8 genes, which likely contributed to hypoxic adaptation.

378 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P297 - SEQUENCE ANALYSIS OF 25 AUTOSOMAL STRs INCLUDING SE33 USING AN IN-HOUSE MPS PANEL IN FOUR POPULATIONS Ye-Lim Kwon1,2, Bo min Kim1,2, Eun Young Lee1, Kyoung-Jin Shin1,2

1 Yonsei University College of Medicine, Department of Forensic Medicine, Seoul, Republic of Korea 2 Yonsei University, Brain Korea 21 PLUS Project for Medical Science, Seoul, Republic of Korea

The diversity of autosomal STRs can be increased by identifying sequence variations using massively parallel sequencing (MPS), which is useful in the analysis of degraded DNA and mixture. To adopt this informative data to forensic practice, sequence-based allele frequencies for each autosomal STR should be accumulated for interesting population. Especially, SE33 is known as highly polymorphic STR locus, but a few sequence-based data have been reported. Therefore, in this study, we analyzed 25 autosomal STRs including SE33 using an in-house MPS panel for 350 samples from four populations (African Americans, Caucasians, Hispanics and Koreans). The developed MPS panel simultaneously amplified the 28 markers, consisted of 20 expanded CODIS core loci, five additional autosomal STR loci (D4S2408, D6S1043, Penta E, Penta D and SE33) and three sex typing markers (Amelogenin, DYS391 and Y-M175). The barcoded MPS library was generated by two-step PCR method and sequenced on a MiSeq System. The sequence-based alleles of 25 autosomal STRs were identified from FASTQ files using the STRait Razor v3.0 and concordance of length-based genotypes between capillary electrophoresis (CE) and MPS method was also confirmed. As a result of the MPS analysis, more than 160 unique alleles by sequence were determined in SE33, which showed the highest increase rate in the number of alleles followed by D2S1338, D12S391 and D21S11. The flanking region variations that increased the number of alleles were mainly found in SE33, D7S820, D13S317 and D5S818. In this presentation, we will show detailed sequence structure and gains in the number of alleles by the MPS analysis, and describe population-specific characteristics of sequence-based alleles for the autosomal STRs.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 379 P298 - SEQUENCE DATA OF 31 AUTOSOMAL STR LOCI FROM 498 SPANISH INDIVIDUALS Pedro Barrio1, Pablo Martin1, Alonso Antonio1, Petra Müller2, Martin Bodner2, Burkhard Berger2, Walther Parson2, Bruce Budowle3

1 Instituto Nacional de Toxicología y Ciencias Forenses, Madrid, Las Rozas de Madrid, Spain 2 Medical University of Innsbruck, Institute of Legal Medicine, Innsbruck, Austria 3 University of North Texas Health Science Center, Center for Human Identification, Fort Worth- TX, USA

Short Tandem Repeat (STR) sequence-based allele population data were generated from 498 Spanish individuals. The study was facilitated by use of the Precision ID GlobalFiler™ NGS STR Panel v2. This panel enables amplification of 31 autosomal STR (auSTR) loci: D12S391, D13S317, D8S1179, D21S11, D3S1358, D5S818, D1S1656, D2S1338, vWA, D2S441, D5S2800, D7S820, D16S539, D6S474, D12ATA63, D4S2408, D6S1043, D19S433, D14S1434, CSF1PO, D10S1248, D18S51, D1S1677, D22S1045, D2S1776, D3S4529, FGA, Penta D, Penta E, TH01 and TPOX. Each allele was aligned to the GRCh37 (hg19) sequence and nomenclature designated according to the recommendations of the International Society for Forensic Genetics. For concordance testing concordance, a subset of 221 individuals were typed using PowerPlex Fusion 6C and capillary electrophoresis. For the common loci there was 99.95 % allele concordance. The majority of the auSTR loci displayed sequence due to repeat region sequence variation and/or single nucleotide polymorphisms (SNP) residing in the flanking regions. The observed heterozygosity increased in more than half of the loci due to sequence variation. The combined match probability decreased from by five orders of magnitude from length-based data to sequence-based data for the 20 CODIS core STR loci, and an additional 13 orders of magnitude if for all 31 STR loci. The combined typical paternity index increased from by one order of magnitude with sequence-based data compared with length data. This Spanish population study performed in the framework of the EU-funded DNASEQEX project provides STR sequence-based allele frequencies for forensic casework and supports implementation of massively parallel sequencing (MPS) technology in forensic laboratories.

380 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P299 - SEQUENCE POLYMORPHISMS OF 58 STR LOCI IN A TIBETAN POPULATION USING MASSIVELY PARALLEL SEQUENCING Dan Peng1,2, Riga Wu1,2, Haixia Li1,2, Nana Wang1,2, Hongyu Sun1,2

1 Sun Yat-Sen University, Faculty of Forensic Medicine- Zhongshan School of Medicine, Guangzhou, China 2 Sun Yat-Sen University, Guangdong Province Translational Forensic Medicine Engineering Technology Research Center, Guangzhou, China

Massively parallel sequencing (MPS) has become a promising method for forensic STR typing due to its ability to detect a large number of markers and samples simultaneously in a single reaction and provide both length and sequence information of STR alleles. Comparing with traditional capillary electrophoresis (CE) method, the population investigations based on the MPS-STR typing are still insufficient, which limits its full utilization in forensic genetics. In the present study, 58 STRs (27 autosomal STRs, 24 Y-STRs and 7 X-STRs) from 107 unrelated Ti- betan individuals (53 males, 54 females) were sequenced using the ForenSeqTM DNA Signature Prep Kit. Concordance genotypes were obtained with UAS, STRait Razor v2s and STRait Razor v3.0 analysis softwares. A total of 177 more alleles were identified through this sequence-based (SB) approaches than length-based (LB) calling approaches, with the increasing percentages of 41.65 %. Seventeen autosomal STRs, 9 Y-STRs and 5 X-STRs were shown to have increase in allele number, DYF378S1, D12S391, and D2S1338 were the three loci with the highest increasing percentages of 216.67 %, 163.64 % and 136.36 %, respectively. The sequence variants included the repeat region variants only (RRVO), flanking region variants only (FRVO) and repeat region plus flanking region variants (RRFR). A total of 39 novel sequence variations were observed in 20 loci. Twenty-five SNPs and two InDels in 21 loci were also determined in the flanking region. Genetic comparisons with 7 other populations were performed. The total discrimination power (TDP), cumulative probability of exclusion (CPE) and haplotype diversity (HD) were all higher than those from CE methods.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 381 P300 - SEQUENCE VARIATION AND POPULATION CHARACTERISTICS OF THE NEW ZEALAND POPULATION USING THE FORENSEQ™ DNA SIGNATURE PREP KIT Sallyann Harbison1, Ryan England2,3, Andrew Sarman2, Janet Stacey1

1 Institute of Environmental Science and Research Ltd, Forensic Biology, Auckland, New Zealand 2 Institute of Environmental Science and Research Ltd, Forensic Research, Auckland, New Zealand 3 University of Auckland, Forensic Science Program, School of Chemical Sciences, Auckland, New Zealand

The New Zealand population is a diverse one comprised of an indigenous Maori population and more recent immigrants comprising mostly Caucasian people of Western European origin. Much of the publically available allele frequency information and tools for addressing biogeographical ancestry determination are lacking data representing our population. In particular, this includes data from the Pacific Islands for example Samoa, Tonga and New Zealand Maori. This means that we don’t know whether the existing SNPs used in forensic multiplexes are useful to us and we have limited access to existing frequency information. In order to use biogeographical ancestry information effectively we have sequenced over 500 individuals from our population using the ForenSeq™ DNA Signature Prep Kit. We have carried out a variety of statistical tests to investigate the structure of our population to determine how well the ancestry and phenotype informative SNPs behave in New Zealand. We include examples of how we intend to report this information to Police Investigators.

382 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P301 - SEQUENCE‐BASED SAUDI POPULATION DATA FOR THE SE33 LOCUS Hussain Alsafiah1, Yahya Khubrani2, Hadi Sibte1, William Goodwin1

1 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom 2 University of Leicester, Department of Genetics and Genome Biology, Leicester, United Kingdom

Massively Parallel Sequencing systems are now being adopted in many forensic laboratories allowing detailed sequence data for tested samples. By utilising the MiSeq FGx platform and ForenSeqTM Universal Analysis Software (UAS), ForenSeqTM DNA Signature Prep kit simultaneously targets 27 autosomal STRs along with other commonly used markers (Y-STRs, X-STRs, and SNPs). An additional five STRs, including SE33 locus, are included in the PCR mixes, but they are not reported by the UAS. SE33 is the most polymorphic STR, which makes it valuable for forensic applications. Previous studies have demonstrated that by sequencing the SE33 presents 250 % more alleles compared to CE systems. A set of 87 reference samples from Saudi population, which were already profiled with Global- Filer® PCR amplification kit, were sequenced using ForenSeqTM kit on a MiSeq FGx. The data of the SE33 locus were pulled out from the FASTQ files by the STRait Razor v2.0 software after modifying the configuration file. All sequences with ≥ 10 reads were included and the genotypes with ≥ 20 % of allele coverage ratio (ACR) were considered as true genotypes. The SE33 sequences of 87 samples were recovered, 85 of which were within the designated limits, while two samples showed 18 % ACR. The number of SE33 alleles were 69 (130 % increase compared to CE system, 30 alleles). Only one discordance event was observed where the sequence data showed alleles 19,31.2 while CE data showed alleles 18,31.2. This presentation provides SE33 sequence‐based data for the Saudi population including a concordance study between the GlobalFiler® kit and the ForenSeqTM kit.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 383 P302 - SOCIAL, ETHICAL, AND SCIENTIFIC IMPLICATIONS FOR IDENTIFYING SUSPECTS BY USING FAMILIAL SEARCHES IN DNA DATABASES Robert Janevski1, Ljubica Trajkovska1, Renata Jankova2, Branko Kalkovaliev1

1 Ministry of Interior Affairs of North Macedonia, Forensic Department- Biological investigations and DNA identification Unit, Skopje, Macedonia the former Yugoslav Repu 2 University St. Cyril and Methodius- Medical Faculty, Institute of Forensic Medicine- Criminalistic and Medical Deontology, Skopje, Macedonia the former Yugoslav Repu

In recent years, technology of DNA analysis has revolutionized forensics, thereby becoming a priceless tool in criminal investigations and in the forensic identification process. The establishment of DNA databases has led to the effective connection of the suspects with a crime scene. Familial searches are an important strategy for establishing of relationship links through genetic profiles derived from DNA analysis of certain traces found at the crime scene, with any member of the closer family of a potential perpetrator whose DNA profiles may be in the DNA database. This identification of potential relatives can lead to identification of the unknown perpetrator and solving of the criminal event. From a social, ethical and scientific point of view, this investigative strategy has supporters and opponents in the context of its efficiency. This paper aims to review these three aspects and to present the current situation in the Republic of North Macedonia

384 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P303 - START-UP OF THE CRIMINAL GENETIC DATABASE IN MENDOZA, ARGENTINA Laura Locarno1, Corradi Corradi1, Miguel Marino1

1 Public Ministry of Mendoza, Registro Provincial de Huellas Genéticas Digitalizadas, Mendoza, Argentina

The usefulness of DNA databases is widely known and demonstrated. After the successful experiences of the UK and the USA the creation of databases increased rapidly around the world. In Latin America this was slower and more problematic, with Chile and Uruguay being the first to implement them. Argentina was not out of that, indeed, the problems were greater and per- sistent. Although the lack of legislation or applicable laws is a generalized problem, the most difficult one to overcome was the lack of decision, interest and resources by those responsible at an institutional level. In 2016, Mendoza province modifyed its database law by creating the “Registro Provincial de Huellas Genéticas Digitalizadas” which allowed the process of construction and consolidation to begin. From January 2017 all prisoners, convicted and imputed of all types of crimes began to be sampled. This made the database to grow rapidly, reaching 13.821 samples in that year. During 2018, in addition to the daily imputed individuals, it began with the sampling from all the Mendoza Police Department, including the Scientific Police that deals with the crime scene. At present the database has a total of 34.600 individuals. In August 2018, the FBI’s CODIS system was installed, and later the data loading process began. In 6 months we have 65 match or hits of which 36 correspond to sexual assault, 15 to robbery, 10 to homicides and the rest to other cases. Given that within the causes of sexual abuse were able to identify several serial sexual abusers, the 64 hits allowed to clarify 125 criminal cases. These results reaffirm the potential of the databases and give a light of hope for victims of crime. In this work we present the advances and challenges that we face in a chronological order.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 385 P304 - STUDY OF GENETIC MARKERS WITH MEDICO‑LEGAL AND FORENSIC INTEREST IN LISBON’S POPULATION (PRELIMINARY RESULTS) Diogo Rodrigues1,2, Cláudia Vieira Da Silva2, Heloísa Afonso Costa2,3, Maria João Porto2, Eugénia Cunha2,4, Francisco Corte Real5,6, António Amorim2,7,8

1 Instituto de Higiene e Medicina Tropical, Biomedical Scienses, Lisboa, Portugal 2 Instituto Nacional de Medicina Legal e Ciências Forenses, Forensic Genetics, Lisboa, Portugal 3 Faculdade de Ciências da Saúde da Universidade da Beira Interior, Biomedical Scienses, Covilhã, Portugal 4 Faculdade de Ciências e Tecnologia da Universidade de Coimbra, Biology, Coimbra, Portugal 5 Instituto Nacional de Medicina Legal e Ciências Forenses, Presidency, Coimbra, Portugal 6 Faculdade de Medicina da Universidade de Coimbra, Legal Medicine, Coimbra, Portugal 7 Escola de Ciências e Tecnologia da Universidade de Évora, Biology, Évora, Portugal 8 Faculdade de Ciências da Universidade de Lisboa, Genetics, Lisboa, Portugal

We can define a genetic profile, within the scope of medico-legal and forensic genetics, as a description of information relating to a set of different genome locations of an individual, which may take the form of a specific sequence of nucleotides or a numerical form. Nowadays, in accordance with international recommendations, and particularly according to the European DNA Profiling Group (EDNAP), the genetic markers that, within forensics genetics, allow to obtain individualizing genetic profiles, are repetitive sequences of DNA, called STR. There are some variations regarding the minimum number and STR to define a genetic profile. Across Portuguese laboratories of forensic genetics, the 13 STR’s from the CODIS system and at least 10 more STR are analyzed. Nevertheless, in complex cases it could be necessary the study of additional genetic markers. Given the lack of population studies regarding 9 new STR - D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D21S2055 -, it’s necessary to study their frequencies in Lisbon’s population. In this study we analyzed samples from 200 individuals comprised in forensic investigations in progress at Instituto Nacional de Medicina Legal e Ciências Forenses (INMLCF). The samples for this study were randomly selected from both genders and from unrelated individuals without restriction regarding their ages. The obtained results for forensic statistical parameters such as observed heterozygosity, polymorphism information content, power of discrimination and mean exclusion chance, based on single allele frequencies reveal that this system is highly informative and can represent an important tool for genetic identification purposes for forensic investigations ongoing on INMLCF, related to individuals from Lisboa.

386 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P305 - STUDY OF INSERTION-DELETION POLYMORPHISMS (INDELS) IN THE UAE POPULATION Maryam Almheiri1,2, Shaikha Sanqoor2, Mahdi Haidar3, Muhammad Nazir4, Ranjit Vijayan1

1 College of Science, UAE University, AlAin, United Arab Emirates 2 General Department of Forensic Science and Criminology, Dubai Police, Dubai, United Arab Emirates 3 Centre for Forensic Science, University of Strathclyde, Glasgow, United Kingdom 4 College of Biotechnology, University of Modern Sciences, Dubai, United Arab Emirates

Insertion–deletion polymorphisms (indels) are short length diallelic polymorphisms caused by the insertion or deletion of several nucleotide bases. Recently, insertion-deletion polymorphisms have become an important topic in forensic science as they can be used as a supporting tool to short tandem repeats (STRs) typing. In this study, 30 biallelic autosomal indels and amelogenin markers were investigated in 500 individuals in the UAE population using Qiagen Investi- gator DIPplex kit. The allelic frequencies of the short allele of the 30 indel loci were in the range of 0.266 – 0.658. The power of discrimination was observed ranging from 0.550 (HLD56 locus) to 0.655 (HLD101 locus) and the probability of exclusion was found in the range of 0.102 (HLD97 locus) to 0.202 (HLD83 locus). The combined power of discrimination, power of exclusion and matching probability for 30 loci in the UAE population were 0.999999999999754, 0.9941 and 2.46 X 10-13 respectively. This study showed that the Investigator DIPplex kit is a powerful tool to investigate genetic polymorphism in UAE population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 387 P306 - THE EUROFORGEN NAME AMPLISEQ CUSTOM PANEL: A SECOND TIER PANEL DEVELOPED FOR DIFFERENTIATION OF INDIVIDUALS FROM THE MIDDLE EAST/NORTH AFRICA Ditte Truelsen1, Vania Pereira1, Christopher Phillips2, Niels Morling1, Claus Børsting1

1 University of Copenhagen, Department of Forensic Medicine, Section of Forensic Genetics, Copenhagen, Denmark 2 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic Genetics Unit, Santiago de Compostela, Spain

The interest in, and use of, ancestry informative markers in both forensic and clinical genetics is increasing. Several panels have been designed to distinguish individuals from major geographical regions. However, second-tier ancestry panels for better differentiation of closely related populations are needed. The EUROFORGEN North Africa and Middle East ancestry panel (in short, the NAME panel) is a second tier panel designed for differentiating Middle Eastern/North African populations from other population groups. The first version of the panel was developed for the MassARRAY® System (Agena Bioscience). The disadvantages of this method are high DNA input requirements and relatively low multiplexing capacity. Therefore, an Ion AmpliSeq™ custom panel (Thermo Fisher Scientific) was designed to sequence populations of interest using the Ion S5™ (Thermo Fisher Scientific). Of the 111 initially selected SNPs, 107 were successfully incorporated in the AmpliSeq™ multiplex PCR. More than 1,000 individuals from 14 different countries in the Middle East, North Africa, and Europe were typed. The performance of the sequencing assay was assessed by calculating relative locus balances, heterozygote balances, and noise levels. Criteria for the downstream analysis were selected, and genotyping concordance with MassARRAY® results was determined. The same samples were previously typed with the Precision ID Ancestry panel. The combined strength of the NAME panel and the Precision ID Ancestry panel was evaluated.

388 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P308 - THE IMPACT OF THE PRÜM TREATY ON THE PORTUGUESE FORENSIC DNA DATABASE – A BRIEF REVIEW Pedro Brito1, Ana Margarida Bento1, Nair Gouveia1, Lisa Sampaio1, Filipa Balsa1, Virgínia Lopes1, Marta São Bento1, Patrícia Cunha1, Armando Serra1, Maria João Porto1

1 National Institute of Legal Medicine and Forensic Sciences- I.P., Forensics Genetic and Biology Service- Central Delegation, Coimbra, Portugal

The Treaty of Prüm signed on 27 May 2005, by seven Member States of European Union (EU), sought to improve cross-border security cooperation in combating terrorism, cross-border crime and illegal immigration. For this purpose, provisions were set for automated exchanges of data regarding DNA, fingerprints and vehicle registration plates. Later, other Member States expressed their intention to accede to the Prüm Convention, including Portugal. Portuguese forensic DNA profiles database was implemented in 2010, although only in 2015 Portugal started the automated DNA data exchange with other countries. From that time until now, it has connected to a large number of countries in order to fulfil the purpose for which it was intended when accessed the Prüm Convention, following the guidelines of EU De- cision 2008/615/JHA. It is currently exchanging DNA data automatically with 21 Member States. The objective of this work is to describe the evolution of the Portuguese forensic DNA database under Prüm Treaty over the past few years, analysing several information such as the number and type of genetic profiles shared, the number of hits that occurred with other countries, as well as other relevant information.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 389 P309 - THE MATERNAL INHERITANCE OF THE ASHANINKA NATIVE GROUP FROM PERU Filipa Simão1, Catarina Xavier2, Dean Herman Tineo3, Elizeu Fagundes de Carvalho1, Walther Parson2, Leonor Gusmão1

1 Laboratório de Diagnóstico por DNA, State University of Rio de Janeiro, Rio de Janeiro, Brazil 2 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 3 Instituto de Medicina Legal, Universidad Nacional Mayor de San Marcos, Lima, Peru

In the construction of forensic genetic databases, it is important to evaluate intra-population genetic diversity as well as relationships among neighbouring groups. The Amazonia rainforest, in South America, harbours native populations with high ethnic diversity. Thus, the evaluation of the genetic composition of populations in this region represents a challenge, and few studies are available describing its native groups. In this work, the maternal inheritance of 170 Ashanin- ka individuals, living in the Amazonia region of Pasco department, Peru, was evaluated by mtD- NA control region sequencing. As previously observed for other native groups from Amazonia, a low haplotype diversity was obtained, and only native American haplogroups (A2, B2, B4b, C1 and D1) were found. Strong founder effects were observed, especially for sub haplogroups A2aa, B2b+152, C1b and D1, showing a high number of shared haplotypes. Thus, during the Eu- ropean colonial period, the Ashaninka population seems to have remained relatively isolated, which can be explained by its remote location in the tropical forest. A comparison with other native South American populations, from different linguistic families, showed a lack of geographic or linguistic affiliations, highlighting the importance of having specific mtDNA database for the different native groups in South America.

390 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P310 - THE MOST FREQUENT AUTOSOMAL STRs (POWERPLEX FUSION) INVOLVED IN EXCLUSION OF PATERNITY CASES IN A POPULATION FROM SOUTHEAST, MEXICO Paola Nicte Lopez Gonzalez1, Jorge Enrique Bautista-Gonzalez1, Maria Jose Lopez-Gonzalez1, Javier Enrique Sosa-Escalante1, Lizbeth Gonzalez-Herrera2

1 DIMYGEN Laboratorio, Human Identification, Merida, Mexico 2 Center for Regional Research, Genetic Laboratory, Merida, Mexico

Introduction: Short tandem repeats (STR) are very widespread in the human genome; being a source of polymorphic markers, used for the determination of kindship. Few studies indicate the frequency of autosomal STR involved in the exclusion of paternity, as well as, those with a higher percentage of discordance. Aim: To determine the distribution and frequency of the main autosomal STR markers involved in the exclusion of paternity cases in an admixed population from Southeast, Mexico (Yucatan Peninsula). Material and Methods: 78 cases of excluded paternity, were included in the study: 54 trios and 24 motherless. All subjects belong to an admixed population with Mayan ethnicity from Southeast of Mexico. PowerPlex Fusion System (PPFS) was used for genotyping. The distribution and frequency of the autosomal STRs involved in each case of paternity exclusion were recorded based on 22 autosomal STR of PPFS. Results: The number of STR which determined the exclusion in all cases ranged from 5 to 18 markers, being up to 14 STR for motherless cases. Paternity exclusions occurred most frequently with 12-STRs (22.22 %) for trios and 9-STRs (37.5 %) for motherless cases. The autosomal STR with the highest percentage of discordance is PENTA-E, for all cases 73.08 %, 85.19 % for trios. For motherless cases were D1S1656 and PENTA-E (45.83 %, both). Conclusions: The most frequent number of autosomal STR marker to confer a paternity exclusion was 12, the most informative STR marker for exclusions was PENTA-E.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 391 P311 - THE PHYLOGENETIC ANALYSIS OF TWO ETHNIC GROUPS LIVING IN THE LÂM ĐỒNG PROVINCE, VIETNAM, BASED ON Y CHROMOSOMAL STR LOCI Grażyna Zielinska1, Duc Hung Nguyen2, Romain Gastineau3, Maria Szargut1, Sandra Cytacka1, Joanna Arciszewska1, Katarzyna Jałowińska1, Andrzej Ossowski1

1 Pomeranian Medical University in Szczecin, Department of Forensic Genetics in Szczecin, Szczecin, Poland 2 Saigon University, Faculty of Natural Sciences Pedagogy, Hồ Chí Minh, Viet Nam 3 Faculty of Geosciences, University of Szczecin, Natural Sciences Research and Educational Center and Palaeoceanology Unit, Szczecin, Poland

Population studies from different parts of the world are extremely important for forensic genetics. Their significance in biostatistical calculations is crucial. Genetic identification of unknown persons would not be possible without performing biostatistics calculations. The area of population research is important for identification studies within both current cases and historical studies, for example of victims of armed conflicts from different parts of the world. Vietnam is a country in Southeast Asia. It has an estimated population of 94.6 million inhabitants (2016), distributed among 54 officialy recognized ethnic groups. It is the 15th most populous country in the world. Despite such a large and diverse population, the YHRD database contains a total of only 519 haplotypes from eight different ethnic groups of Vietnam, of which 209 are in the minimal loci range. Our research will focus on analysis of Y chromosome haplotypes of 64 non-related male individuals, originating from two populations from the Vietnamese Central Highlands. This work will complete a database with additional 64 individuals from two additional ethnic groups, analyzed with YFiler Plus set of loci. The first population is the K’ho people, who live in the Lâm Đồng province located in the Central Highlands region of Vietnam. The second population, the Mạ or Maa, is also concentrated mostly in the Lâm Đồng Province of the country, particularly in the area of the upper Đồng Nai River located in the western part of the province. Both populations speak related Mon-Khmer languages that belong to the Bahnaric group.

392 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P312 - THE SPECIFIC HAPLOTYPE OBSERVED IN MONGOLIAN POPULATION Uyanga Ganbold1,2, Purevdulam Sharavdorj3, Oyunchuluun Yadamsuren4, Ganbold Suren3

1 The Mongolian National University of Education, School of Mathematics and Natural Sciences, Ulaanbaatar, Mongolia 2 National Center for Public Health, Environmental and Toxicology Laboratory, Ulaanbaatar, Mongolia 3 The National Institute of Forensic Science of Mongolia, Department of Specialized Examination, Ulaanbaatar, Mongolia 4 The National University of Education, School of Mathematics and Natural science, Ulaanbaatar, Mongolia

In this study we typed 23 short-tandem repeat (STR) loci (DYS576, DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570, DYS635, DYS390, DYS439, DYS392, DYS643, DYS393, DYS458, DYS385, DYS456 and YGATAH4) in the Mongolian population by using PPY23 kit. As a result, a unique haplotype 17–14–20–31–15–11–27–12–12–10– 14–18–21–23–12–12–12–16–15–15,15–15–11 was found. The 16 allele in the DYS393 locus of the 23 Y-STR haplotype data set detected was the first case in the Mongolian population research and it is a rare haplotype not matched to Y-Chromosome Reference Database. Further, we investigate frequency distribution of this specific haplotype.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 393 P313 - TRACING THE GENETIC ORIGINS OF THE MALTESE POPULATION Joanna Vella1,2, Martin Bodner3, Nicole Huber3, Bettina Zimmermann3, Maria Geppert4, Marion Nagy4, Joseph Borg2,5, Alex Felice1,2, Lutz Roewer4, Walther Parson3,6

1 University of Malta, Department of Physiology and Biochemistry, Msida, Malta 2 University of Malta, The Malta BioBank BBMRI.mt- Centre for Molecular Medicine and Biobanking, Msida, Malta 3 Medical University of Innsbruck, Institute of Legal Medicine, Innsbruck, Austria 4 Charité Universitätsmedizin, Institute of Legal Medicine and Forensic Sciences, Berlin, Germany 5 University of Malta, Department of Applied Biomedical Science, Msida, Malta 6 The Pennsylvania State University, Forensic Science Program, Pennsylvania, USA

The Maltese Islands hold a rich demographic history. Historical records trace population origins to the Temple people, however contemporary Maltese are descendants from those who re-populated the islands at the turn of the first Millennium AD. No Maltese lineage marker data is available on EMPOP or YHRD. Two new high-quality datasets were setup to evaluate Maltese lineages: the mitochondrial DNA (mtDNA) control region (CR) and Y-STR chromosome markers. A new random population collection of 800 samples with associated ancestry data is archived in the Malta BioBank (BBMRI.mt). The EMPOP protocol was used to amplify and sequence a subset of 300 samples with a minimum of four EMPOP sequencing primers. mtDNA haplotypes were checked on EMPOP and Phylotree. The PowerPlex® Y23 system was used to analyse 400 unrelated males. NevGen was used to predict Y-STR haplogroups which were confirmed by SNP analysis with HRM The majority of the observed Maltese mtDNAs (77 %) could be attributed to Westeurasian haplogroups: H (35 %), T (18 %), K (12 %), J (5 %), U (5 %), X (1 %), W (1 %). African mtDNA lineages were also present: L1 (0.4 %), L2 (10 %), L3 (1 %), M1 (0.4 %). Y haplogroups show all major Euro- pean Y chromosome clades R1, J2, E1b1b, G, and I. This population genetics research provides a Maltese lineage marker reference for applications in forensic and population genetic studies.

394 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P314 - TRI-ALLELIC PATTERN AT THE DYS385A/B LOCUS Stefania Turrina1, Domenico De Leo1

1 University of Verona, Department of Diagnostics and Public Health, Verona, Italy

Tri-allelic patterns have been rarely described at a single Y-STR locus. Here we report a tri-allelic pattern at the DYS385 locus that has been detected in one male subject during a population study conducted in the North Italy on unrelated males using the PowerPlex Y23 System. At the beginning, the DYS385 locus was genotyped as alleles 13–15 and subsequently this finding was confirmed using the ForenSeq DNA Signature Prep Kit. Although, the MiSeq FGx result was consistent with that generated by the CE system, both systems revealed an unbalanced bi-allelic pattern at DYS385. Based on the patterns and peak area under alleles for CE and the reads for NGS method, the DYS385 allele designation was revised as a tri-allelic patterns 13,13 and 15. In this case, a tri-allelic pattern of type 2-B was identified in which the peak area/ reads of allele 15 was of normal height and the peak area/reads of allele 13 was twofold higher due to the superposition of two identical alleles 13.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 395 P315 - UTILIZATION OF THE CIFS DNA DATABASE TO MONITOR RECIDIVISM Nongnuch Boonderm1, Worawee Waiyawuth1, Suriyanratakorn Danuphol1, Sireethron Sangpueng1, Chalampoo Wongvoravivat1

1 Central Institute of Forensic Science, Ministry of Justice, DNA Division, Bangkok, Thailand

Since 2004, under the integration project of Central Institute of Forensic Science (CIFS) and the Department of Correction, Ministry of Justice, CIFS has collected biological samples from prisoners who will be released within four months to one year with all offences. One aspect of this project is to prevent and monitor detained prisoners from repeated offences. Until March 2019, buccal cells on FTA paper were collected and analyzed more than 120,000 samples, then were uploaded to the CIFS DNA database. Presently, CIFS DNA Database has reached approximately 180,000 profiles, which consists of DNA profiles from crime stain samples, individuals who related to crimes and prisoners roughly amount to 16,000, 48,000 and 120,000 respectively. Mainly autosomal STR markers were collected in the DNA database. Y-STR markers are also analyzed and uploaded, especially crime stains from sexual assault cases and their potential suspects. Approximately 2,000 hits were reported on various offenses such as explosive, unrest, homicide, sexual assault, and drug smuggling. Interestingly, 147 prisoners were found matching against samples from drug smuggling (66), organized crimes (26), and others (35). This number demonstrated that detainers trend to commit crimes again or recidivism. Consequently, the DNA database can be applied to monitor repeated offenders. To expand the capabilities of DNA investigations, in the near future, CIFS plans such as collecting newly registered prisoners and all prisoners in Thailand in order to monitor repeated offences and perhaps increase the hit rate. However, this report is only an observation, which will not be any intention to point out that prisoners will commit a new crime again.

396 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P316 - VALIDATION OF A POPULATION SIMULATION MODEL FOR THE ESTIMATION OF Y-HAPLOTYPE FREQUENCIES IN FORENSIC CASES USING A LARGE FRENCH-CANADIAN DATASET Roxane Landry1, Mikkel Meyer Andersen2, Emmanuel Milot1

1 Université du Québec à Trois-Rivières, Departement of chemistry, biochemistry and physic, Trois-Rivières, Canada 2 Aalborg University, Department of Mathematical Sciences, Aalborg, Denmark

In forensic cases where a DNA mixture of a female major and a male minor contributor (e.g. on intimate swabs taken after sexual assaults) is found, the analysis of Y chromosome by STR typing can help to separate these contributors. However, if a match occurs between a Y-STR profile recovered from a crime scene and that of a suspect, a probative value is hard to establish principally due to paternal relatedness of Y chromosomes and to the lack of an adequate Y database. The objective of our study is to use a large dataset of the French-Canadian population (from Quebec, Canada) to test a simulation model that proposed a new and promising model to calculate the probative value of a Y haplotype. It consists of a population simulation model and software to approximate the distribution of the number of males with a matching Y profile. The dataset that we possess is composed of genealogical data of Quebec’s population since its foundation in 1608 as well as molecular information (Y-STR profiles) for about 300,000 of men who lived in different period of time. This large dataset allowed us to validate the proposed simulation model and to test the impact of population structures such as founder effect and genetic drift on the accuracy of the model. Our preliminary results show that some haplotypes are largely frequent in the population suggesting that the founder effect has an impact and need to be taken into account. Further analyses are in progress to understand more finely that impact, and if the simulation method can be extended to take into account the founder effect in a population. Ultimately, others population structure parameters will be tested, such as genetic drift and the addition of new Y haplotypes in the contemporary population by migrants.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 397 P317 - VALIDATION OF THE MICROREADERTM 28A ID KIT: A 28 LOCI SYSTEM FOR FORENSIC APPLICATION Jing Zhu1, Yifan Li2, Chuguang Chen2, Shengqiu Qu1, Yuqing Liu1, Lin Zhang1, Shuqiang Cao1, Weibo Liang1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Beijing Microread Genetics Co. Ltd, Beijing Microread Genetics Co. Ltd, Beijing, China

Short tandem repeat (STR) analysis is widely used in forensic DNA casework and database applications. The rapid growth of DNA databases reminder that more markers are required in multiplex kits to provide more information in forensic science. With the current capillary electrophoresis (CE) technology, a kit which involves autosomal STR loci and several Y chromosome loci can provide more information compared with only autosomal STR loci. These commercial STR kits are needed to be developed to provide more information, e.g., higher power of discrimination (PD) and probability of exclusion (PE) value, to facilitate various forensic cases. The MicroreaderTM 28A ID kit (Beijing Microread Genetics Co. Ltd, China) is a 6-dye system, which could amplify 25 STR loci (including 20 CODIS Core Loci), 2 Y-Indels and the amelogenin locus in one reaction. And it can be amplified using the filter paper or FTA card directly, without DNA extraction and purification. The validation of MicroreaderTM 28A ID kit was performed according to the guidelines of “Validation Guidelines for DNA Analysis Methods (2016)” described by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-based studies, sensitivity study, precision and accuracy evaluation, stutter percentage and peak height ratio, inhibitors, species specificity and DNA mixture studies. The results suggested that MicroreaderTM 28A ID kit is a robust and reliable amplification kit which can be used widely for forensic purposes.

398 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P318 - VALIDATION OF Y-ANCESTOR TIME CALCULATORS FOR FORENSIC FAMILIAL SEARCHING Sofie Claerhout1, Charlotte Defraye1, Ronny Decorte1,2

1 KU Leuven, Forensic Biomedical Sciences, Department of Imaging and Pathology, Leuven, Belgium 2 University Hospital Leuven, Department of Forensic Medicine, Laboratory of Forensic Genetics and Molecular Archaeology, Leuven, Belgium

When no suspects are identified in a forensic case as the donor of a biological trace, familial searching through the Y-chromosome can be used as an alternative identification method to find paternally related males of the perpetrator. When a relative is identified with a close Y-haplotype match compared to the evidence sample, the time to their most recent common ancestor (tMRCA) can be estimated. This information could be of interest for forensic investigators in order to reconstruct their genealogy. To date, different online tMRCA calculators exist based on the standard infinite allele model (IAM). The aim of this study was to validate three most commonly used online tMRCA calculators (McDonald, Walker and McGee) by comparing them to our genetic genealogy database containing 1,126 genealogical pairs with known tMRCA, in which 42 Y-STR markers were analyzed. Based on this validation, we observed that the McGee calculator is very discordant with our dataset, while the others give quite similar results. Over- all, the tMRCAs from our genealogical pairs are located within the 95 % CI of the calculators. However, the tMRCA is slightly underestimated for pairs with a close Y-haplotype match, and overestimated when more Y-STR differences are present. When haplogroup specific mutation rates were taken into account, they consistently underestimated the tMRCA. It can be concluded that these calculators were not appropriate for use in familial searching due to their large 95 % CI. To further improve tMRCA estimations, a novel user-friendly tMRCA calculator should be developed including individual Y-STR mutation rates and Y-STR allele calls to take possible mutation characteristics into account, like multistep or parallel Y-STR modifications, which can cause divergent results.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 399 P319 - WEIGHING SUBSTRUCTURE IN ARGENTINA CONSIDERING DIFFERENT POPULATION CLUSTERS Maria Gabriela Garcia1, Cecilia Catanesi2,3, Gustavo Penacino4,5, Nádia Pinto6,7,8

1 Laboratorio MANLAB, Área de Filiaciones, Buenos Aires, Argentina 2 Instituto Multidisciplinario de Biología Celular IMBICE, Laboratorio de Diversidad Genética, Buenos Aires, Argentina 3 Universidad Nacional de La Plata, Facultad de Ciencias Naturales y Museo, Buenos Aires, Argentina 4 Colegio Oficial de Farmacéuticos y Bioquímicos, Colegio Oficial de Farmacéuticos y Bioquímicos, Buenos Aires, Argentina 5 Fundación INGEN, Fundación INGEN, Monte Grande, Argentina 6 Institute of Pathology and Molecular Immunology from University of Porto IPATIMUP, Population Genetics and Evolution, Porto, Portugal 7 Instituto de Investigação e Inovação em Saúde- I3S- Universidade do Porto, Instituto de Investigação e Inovação em Saúde- I3S- Universidade do Porto, Porto, Portugal 8 Centro de Matemática da Universidade do Porto, Centro de Matemática da Universidade do Porto, Porto, Portugal

Population representative databases are necessary for the quantification of the genetic evidence in forensic casework. It is thus essential to perform a correct clustering of the samples, as genetic distances could substantially vary using different regional patterns. Consequently, the final clustering scheme for haplotype frequencies and estimates of forensic parameters can also change. In this study 914 unrelated haplotypes from residents of all Argentina, typed for a set of 12 X-STRs, were considered. To compute differentiation analyses and HWE estimations we considered five alternative divisions of Argentine territory: i) North, Center, South, ii) Buenos Aires, Center, Novo Cuyo, Norte Grande and Patagonia, iii) Northwest, Northeast, Pampeana, Cuyo and Patagonia, iv) North- west, Northeast, Center, South, v) Northwest, Northeast, Middlenorth, Center and South, using the same samples grouped according to each of the six scenarios. Evidences of stratification were not found for four of the clustering patterns considered (i to iv), contrasting to what was obtained for the last division (v). Our work shows that an inadequate clustering of the samples can lead to misleading results concerning population stratification and thus multiple clustering alternatives of the same territory should be considered to ensure sound conclusions. This analysis is particularly pertinent in populations, such as Argentine, where clime, geography and demography deeply vary across the territory, which may result in non-coincident administrative and natural divisions.

400 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P320 - X-CHROMOSOME ANALYSIS IN AN UNUSUAL DEFICIENCY MATERNITY CASE Ciro Di Nunzio1, Michele Di Nunzio2,3, Flavio Saia1, Aldo Di Nunzio1, Pietrantonio Ricci4, Silvano Presciuttini5

1 Magna Graecia University, Medicine Faculty, Legal Medicine- Forensic Genetics Laboratory, Germaneto CZ, Italy 2 Barcelona University- Faculty of Medicine, Public Health, Barcelona, Spain 3 Biogem Scarl, Genetics Research, Forensic Genetics Laboratory, Ariano Irpino AV, Italy 4 Magna Graecia University, Medicine Faculty, Legal Medicine, Germaneto CZ, Italy 5 Pisa University- Faculty of Medicine, Translational Research and New Technologies, Pisa, Italy

The X-chromosome markers are more efficient than autosomes in special cases of kinship analysis. Here, we show an unusual case, where two half-sisters (AR, AP), daughters of the same woman AL, wanted to know whether they were also half-sisters of two other women (MM, MS) and a man (CR). These latter subjects were known to be children of different fathers. DNA was available from AR’s and AP’s maternal aunt AA. Two hypotheses were investigated: 1) AR, AP, MM, MS and CR were children of the same mother (AL) and five different fathers; 2) MM, MS and CR were unrelated to AR and AP. The genotypes were obtained by the AmpFlSTR® Iden- tifiler® and Investigator® ArgusX12 kits. Using the undisputed relationships (AA being aunt of the half-sisters AR and AP), the X-haplotypes of AR’s and AP’s maternal grandparents could be inferred. Grandmother’s X-haplotypes were also shared by MM, MS and CR, thus supporting hypothesis 1. A Mendelian inconsistency was observed at locus DXS10134, though it could not be distinguished between mutation and intra cluster recombination. Extra cluster meiotic recombination events were also observed, as some other interesting outcomes useful for forensic genetics discussion. According to ISFG guidelines the biostatistical evaluation in kinship analysis was based on a likelihood ratio approach, using FamiLinkX and Familias 3 programs respectively on the X-chromosome and autosomes analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 401 P321 - X-STR DECAPLEX STUDY ON THE POPULATION OF IMBABURA-ECUADOR Ana Karina Zambrano1, Mishelle Vaca2, Margarita Vela2, Gisella Fiallos2, Carmen Gruezo2, Cristina Rodríguez2, Juan José Builes3, Anibal Gaviria2

1 Centro de Investigación Genética y Genómica, Facultad Ciencias de la Salud Eugenio Espejo-Universidad UTE, Quito, Ecuador 2 Laboratorio de Genética, Centros Médicos Especializados Cruz Roja Ecuatoriana, Quito, Ecuador 3 GENES Ltda, Laboratorio del Genética Forense y Huellas Digitales del DNA, Medellín, Colombia

X chromosomal markers provide data of paternal and maternal lineages, which, together with their characteristics of inheritance and recombination, represent a useful tool in population genetics and as a complement in forensic and kinship analysis. The multiplex system was used for the analysis of 100 unrelated individuals from Imbabura-Ecuador who had signed the informed consent for genetic studies. Allele frequencies and parameters of population interest were calculated for each X-STR. The most informative marker was the DXS6809 and the lowest the DXS7133. High discrimination power values were obtained for men and women (greater than 99.99 %), as well as high mean exclusion chance in trios (mother / daughter / father) 0.9999948011 and duos (father / daughter) 0.9992880836, parameters that confirmed the potential of these 10 X -STRs. Genetic distance (FST) distances between the present study and other available studies from Pichincha and Ecuador were performed, and those revealed no significant differences. Internationally, the comparisons shown greater distance with Rio de Janeiro, Cantabria and Portugal, and similarities with Bogotá-Colombia.

402 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P322 - Y CHROMOSOME SEQUENCE VARIATION OF COMMON FORENSIC STR MARKERS AND THEIR FLANKING REGIONS AMONG POLISH POPULATION Maria Wróbel1, Agnieszka Parys-Proszek1, Magdalena Marcińska1, Tomasz Kupiec1

1 Prof. Dr. Jan Sehn Institute of Forensic Research, Forensic Genetic Section, Kraków, Poland

Analysis of Y-chromosome STR markers is a standard procedure with forensic examination of sexual assault cases, in paternity/kinship cases and identification of human remains. Massively parallel sequencing (MPS) technology proves to be reliable method in analysis of DNA mixtures or low quality of DNA samples, result provides high degree of uniqueness of the profiles. In cases of frequent Y-STR haplotypes, sequence analysis of an allele and flanking region gives a chance to distinguish microhaplotypes of the samples. The purpose of this study was to investigate DNA sequence variability within the Y-STR markers in polish population. Two groups of samples were examined: population group which included reference samples from Poland and challenging sample group consisting of male bone samples with different stages of degradation. MPS was carried on with Illumina’s ForenSeq kit on the MiSeq FGx instrument. Samples were analyzed according to the thresholds set during internal validation. Final step was the estimation of variability’s frequency. Revealed microhaplotypes and their frequency differ between markers. Some of the analyzed sequences showed no variability in the analyzed set of samples. Confronting the population data with the case samples showed that this type of information can significantly improve kinship calculation and mixture analysis. Integrating Y-STR and Y-SNP data provide information which can increase discrimination power of the results. Moreover, Y-SNP analysis allow obtaining more relevant information in low-quantity and low-quality, degraded samples. MPS technology offers an opportunity to provide significant amount of genetic information and, as a consequence, strengthening the statistical power of the evidence of a match or a kinship identification.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 403 P323 - Y-CHROMOSOMAL HAPLOTYPE DIVERSITY FOR 27 STR LOCI IN THE TIGRAY POPULATION (NORTHERN ETHIOPIA) Kiros Haddish1, Hareesh Kumar1, Sara Raddi2, Elena Chierto2, Giancarlo Di Vella2, Agajie Likie Bogale3, Eleni Kidane3, Ajanaw Yizengaw3, Yimam Getaneh3, Carlo Robino2

1 University of Mekelle, Forensic Medicine, Mekelle, Ethiopia 2 University of Turin, Public Health Sciences and Pediatrics, Turin, Italy 3 Ethiopian Public Health Institute, National HIV Molecular Laboratory, Addis Ababa, Ethiopia

In lineage markers like Y-chromosomal STR (Y-STR) loci, owing to the lack of recombination, the calculation of random match probabilities and kinship indexes requires the use of large population reference databases. Following the migration crisis that affected Southern Europe in the last 10–15 years, the number of forensic DNA tests involving subjects of Eastern African ancestry has dramatically increased. The Horn of Africa is among the main areas of origin of migrants trying to reach Europe through the so-called central Mediterranean route (from Libya to Sicily). Migration-related accidents in the Straits of Sicily are commonplace. In such circumstances, Y-STR analysis can effectively complement autosomal STR data in the identification of shipwreck victims and help reuniting families separated during the crossing. To expand current Y-STR haplotype reference data for Eastern Africa, 250 Ethiopian volunteer donors self-reported as having the paternal grandfather of Tigray origin were analyzed (Me- kelle University Research Ethics Review Committee authorization ERC 0841/2016). The Tigray ethno-linguistic group, which represents over 95 % of the population in the Regional state of Tigray (Northern Ethiopia), also accounts for ~50 % of the population in neighboring Eritrea, one of the top sub-Saharan countries of origin for refugees and unaccompanied minors travelling the central Mediterranean route. The results obtained using the YFiler Plus PCR Amplification kit (Thermo Fisher Scientific) were compared with those available for other Eastern African ethno-linguistic groups and neighbor

populations from Africa and the Middle East by means of pairwise genetic distances (FST), analysis of molecular variance and multidimensional scaling.

404 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P324 - Y-CHROMOSOME STR HAPLOTYPES STUDY IN A POPULATION SAMPLE FROM ARGENTINA Florencia Gagliardi1, Luciana Rabitti1, Nicolás Furman1, Franco Marsico1, Walter Ruben Bozzo1, Mariana Herrera Piñero1

1 Banco Nacional de Datos Genéticos, Ministerio de Educación Cultura Ciencia y Tecnología, Buenos Aires, Argentina

Object: We studied the distribution of Y-chromosome STRs in a population sample from Argen- tina and compared it with other Argentinian databases with the purpose of obtaining a more statistically robust database. Materials and methods: DNA was extracted from 699 unrelated Argentinian blood samples using an automated QIAcube (Qiagen) system. Y-STR profiles were obtained by using Y filer Plus PCR Amplification kit (Applied Biosystems), run by capillary electrophoresis with a 3500 Genetic Analyzer (Applied Biosystems) and analyzed with GeneMapper ID-X software. Haplotypes were grouped in 7 regions defined by the province of the donors’ current place of residence. Arlequin V3.5.2.2 software was used for Haplotype diversity and fixation index (Fst) estimations in order to analyze haplotype distribution and population substructure in the sample. Results: 695 different haplotypes were found. 691 were unique haplotypes and 4 were shared between pairs of 2 individuals. The haplotype diversity was 0.9986. Fst test showed no population substructure. Additionally, we compared our population sample with those published by Toscanini et al. (2015) and Purps et al. (2014) and no significant differences were found. Discussion: Due to the fact that there are no significant differences between our sample and those published, we are able to merge our data with them and get a more representative and updated database of Argentinean population.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 405 DNA TYPING METHODOLOGIES AND STRATEGIES

P325 - “UNUSUAL” TISSUES AND SAMPLE COLLECTION STRATEGIES ON EXHUMED BODIES Vincenzo Agostini1, Sarah Gino2, Serena Inturri3, Alberto Marino4, Nicola Staiti4, Maurizio Sticchi4, Enrica Chiti5, Pasquale Linarello6, Guendalina Gentile7, Paola Primignani8, Maddalena Giriodi9, Paolo Bailo8, Andrea Piccinini8

1 Università del Piemonte Orientale, DiSIT, Alessandria, Italy 2 Università del Piemonte Orientale, Department of Health Sciences, Novara, Italy 3 Università degli Studi di Torino, Dipartimento di Scienze della Sanità Pubblica e Pediatriche- Laboratorio di Scienze Criminalistiche “Carlo Torre”- Sezione di Genetica Forense, Torino, Italy 4 Reparto Carabinieri Investigazioni Scientifiche, Sezione Biologia, Parma, Italy 5 Istituto Scienze per la Vita, Scuola Superiore Sant’Anna, Pisa, Italy 6 Eurofins Genoma Group, Forensic Unit, Milano, Italy 7 Università degli Studi di Milano, Forensic Histopathology Laboratory, Department of Biomedical Sciences for Health, Milano, Italy 8 Università degli Studi di Milano, Forensic Genetics Laboratory, Department of Biomedical Sciences for Health, Milano, Italy 9 Università degli Studi di Milano, Department of Biomedical Sciences for Health, Milano, Italy

The choice of soft or hard tissues to be sampled in case of exhumation of corpses for identification purposes or family relationship testingis made on the basis of the degradation conditions of the corpse: the more the corpse is degraded, the less DNA is expected to be retrieved from soft tissue. In these “difficult” cases, the choice of the “best” tissue samples usually falls on teeth and bones, even though the DNA extraction procedure requires time and effort and it can often result in negative results. We here present the results of a daily practice survey that shows that it is possible to obtain good results even on DNA extracted from tissues that appear to be less “appealing” to the examiner by performing “simple” corneal/scleral swabs and collecting cartilage. While DNA extracted from cartilage has been already described, to our knowledge there is no evidence of publications in the scientific literature dealing with cornea/sclera as a source of DNA in the forensic laboratory. The obtained results demonstrate that it may be advisable to consider other tissues which bear the potential of returning good profile results despite not appearing particularly useful. A better control of contamination is also a positive issue to consider due to the less complex extraction procedures employed when using soft rather hard tissues.

406 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P326 - A MODIFIED DIRECT PCR AMPLIFICATION USING THE GLOBALFILER™ PCR AMPLIFICATION KIT ON BLOODSTAINS COLLECTED USING MICROFLOQ™ DIRECT SWABS Wai Yin Chong1, Yongxun Wong1, Boon Kiat Ng1, Afiqah Razanah Rosli1, Holden Lim1, Jacquelyn Tay1, Christopher Kiu Choong Syn1, Xavier Chan1

1 Health Sciences Authority, Biology Division- DNA Profiling Laboratory, Singapore, Singapore

Our Laboratory recently adopted a direct PCR amplification method for rapid DNA profiling of bloodstains collected with microFLOQ™ Direct swabs. The direct PCR amplification method utilizes samples collected on the microFLOQ™ Direct swabs as a template for PCR amplification, by-passing the DNA extraction and quantification steps in a standard DNA profiling workflow. Without DNA extraction, there is no liquid DNA generated and repeat PCR amplifications is not possible. To circumvent this possible limitation of a direct PCR amplification method, modifications were made to the sample collection step using the microFLOQ™ Direct swabs to yield a DNA lysate which could then be used for two independent PCR amplifications. Several modified direct PCR amplification methods were compared against the original direct PCR amplification method on bloodstains, ranging from varying the mode of sample collection to the number of swabs used for PCR amplification. The optimised method was subsequently assessed on various bloodstained substrates commonly submitted to the Laboratory for DNA profiling. This study shows the results of these comparisons and assessments. In conclusion, the modified direct PCR amplification method allows for two independent PCR amplifications of the same bloodstain to be performed, thus allowing DNA profiles to be verified in a second PCR amplification (if necessary).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 407 P327 - A NEW SET OF DIP-SNP MARKERS FOR DETECTION OF UNBALANCED AND DEGRADED DNA MIXTURES Jinding Liu1, Jiaqi Wang1, Deqing Chen1, Keming Yun1, Jiangwei Yan1, Gengqian Zhang1

1 Shanxi Medical Universiy, School of forensic medicine, Jinzhong, China

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data showed that DIP-SNPs were capable of detecting the minor DNA in unbalanced mixture. The genotyping system was based on allele-specific amplifications of haplotypes formed by a deletion/insertion polymorphism (DIP) and a single nucleotide polymorphism (SNP). In this study, we developed 20 DIP-SNPs which were complement to our prior developed system and improved the system’s capability to solve UDM. The multiplex PCR and SNaPshot assay were established for these DIP-SNPs genotyping in a Chinese Han population based on short amplicons (less than 200 bp). Allele frequencies were estimated based on 60 samples and probability of informative markers (I value) was introduced to estimate the marker efficiency for detecting minor DNA in a mixture. This compound markers has a sensitivity from 1:100 to 1:1000 in a DNA mixture has no gender restriction with 1ng-10ng DNA when detected the minor DNA contributor. The average informative value (I value) of 0.298 meant that the minor contributor in approximately one-third of the mixtures could be detected. In this work, genomic DNA was degraded by incubation at 50 °C. When the autosomal STRs failed for the presence of long fragment markers, all the DIP-SNP markers were successfully typed. In conclusion, the DIP-SNPs could be considered as an alternative marker option, as they may help to unravel the presence of a completely hidden minor contributor that remains undetected when using conventional STR analysis.

408 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P328 - A PILOT STUDY COMPARING EFFICIENCY OF CONVENTIONAL STR TYPING AND MASSIVELY PARALLEL SEQUENCING IN SPANISH CIVIL WAR SKELETAL SAMPLES Miriam Baeta1, Eva Granizo1, Caterina Raffone1,3, Javier Gamboa2, Marian M. De Pancorbo1

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 BIOGENETICS, I+D+i, Vitoria-Gasteiz, Spain 3 Society of Sciences Aranzadi, Physical Anthropology, Donostia, Spain

DNA profiling from skeletal remains often involves a daunting challenge due to the damage and/or degradation of the recovered genetic material. Therefore, nuclear DNA typing can often partially or completely fail, impeding the resolution of identification cases. Up to now, technologies based on capillary electrophoresis (CE)- have been the gold method for nuclear DNA typing. However, CE methods are limited by the number of markers that can be multiplexed together and the different type of markers included in each multiplex (usually one marker type per multiplex). Recently, Massively Parallel Sequencing (MPS) systems, also known as next- gen- eration sequencing, have become a potential tool to overcome some of the afore-mentioned limitations (higher number and different types of markers in one run). Our experience in the genetic analysis of skeletal samples from Spanish Civil War graves using conventional STR (Short Tandem Repeats) typing methods have resulted in less than one third of the analyzed samples providing an non-informative profile (<12 STRs). In these cases, mitochondrial DNA analysis were the method of choice for furthering typing, but it presented the limitation of only allowing the study of maternal lineages. With the aim of increasing the number of nuclear markers successfully typed, a pilot study was carried out comparing the performance of MPS system and conventional STR typing methods. A subset of samples from the Spanish Civil War was analyzed using the ForenSeq™ DNA Signature Prep Kit –MPS system- and CE kits (AmpFLSTR NGM SElect PCR Amplification Kit and PowerPlex® Y23 System). The results support the potential of the MPS system in cases where STR typing partially fails and SNP analysis provides an additional source of genetic information.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 409 P329 - A RARE GENETIC GENDER ANOMALY IDENTIFIED IN A PATERNITY CASE PRESENTING AMEL-Y DROPOUT Renata Alis Nicolae1, Andrea Maria Constantinescu1, Andrei Catalin Costea1, Carmen Monica Constantinescu1, Georgiana Girbea1, Elena Neagu1, Ligia Elena Barbarii1

1 National Institute of Legal Medicine, Genetics, Bucharest, Romania

The examination of different sets of sex markers in view of gender determination may seem a routine and yet, no forensic DNA expert is exempt from dealing with challenging cases on this issue, even if genetic anomalies related to gender occur rarely. We report a AMEL-Y dropout case revealed on the occasion of a routine paternity investigation, which proved to be a rare SRY positive, 46,XX male syndrome, also known as de la Chapelle syndrome. The objective of our case report is to highlight the importance of forensic PCR kits to elucidate anomalies related to gender and avoid sex misinterpretation either in paternity testing or human identification. The subject is a 36-year-old male requesting a paternity DNA test because he was having doubts related to his 2 year-old child born during his marriage. The standard testing results performed on 16 STRs indicated an exclusion from paternity of our male subject which additionaly exhibited an AMEL-Y dropout. We further investigated the male sample using a combination of PCR kits (Y-STRs, X-STRs, Amelogenin and SRY gene) and were able to identify the SRY gene, the loss of the major part of the Y-chromosome, except of a small region surrounding the SRY gene, and also the presence of two X-chromosomes. Thus we finally characterized the AMEL-Y anomaly as being a 46,XX male syndrome. Some basic facts related to the frequency, ethiopathology and diagnosis of the SRY positive, 46,XX male syndrome are reviewed and discussed. Our data indicate that the current forensic PCR kits may elucidate quite complex mechanisms related to Amelogenin abnormalities and overcome the problems related to a wrong genetic gender determination. This study also represents the first reported Amelogenin dropout case detected in the Romanian population.

410 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P330 - A STUDY ON DIRECT PCR AMPLIFICATION USING THE GLOBALFILER™ PCR AMPLIFICATION KIT ON TOUCH AND SALIVA ARTICLES COLLECTED USING MICROFLOQ™ DIRECT SWABS Wai Yin Chong1, Boon Kiat Ng1, Yongxun Wong1, Holden Wei Siong Lim1, Afiqah Razanah Rosli1, Jacquelyn Tay1, Eileen Hui Qi Ng1, Christopher Kiu Choong Syn1

1 Health Sciences Authority, Biology Division- DNA Profiling Laboratory, Singapore, Singapore

We have previously demonstrated that rapid DNA profiling of bloodstains within 3 hours can be achieved through sample collection with microFLOQ™ Direct swabs followed by direct PCR amplification with GlobalFiler™ PCR Amplification Kit and capillary electrophoresis (CE) detection on the ABI 3500xL Genetic Analyser (manuscript submitted). Since the casework exhibits that our laboratory examines frequently include touch and saliva articles, it would be relevant to assess if such a method can be similarly applied. Swabs of touch articles, such as mobile phone screen (n=10), shirt collars (n=9) and watches (n=10) yielded a median allele and profile recovery of 34 alleles, and 85 % of the donor profile, respectively. However, the median allelic peak height was low at 343 Relative Fluorescent Units (RFU). Swabs of saliva-containing articles, such as mouth of drink cans (n=15), mouth and cap of bottles (n=10) and used end of straws (n=9) yielded better results than the touch articles. The median allele recovery of these saliva swabs was 41 alleles, with complete donor profiles obtained. The median allelic peak height obtained from these swabs of saliva-containing articles was 9,040 RFUs. Interestingly, processing of similar areas on such saliva-containing articles using the standard DNA profiling workflow (DNA extraction by Maxwell FSC, quantification, PCR amplification and CE) yielded a median allele and profile recovery of 11 alleles and 28 % of the donor profile, respectively, with a median allelic peak height of only 229 RFUs. In summary, the direct PCR amplification method for samples collected on microFLOQ™ Direct swabs is suitable for the rapid DNA profiling of saliva stains, but may not be appropriate for touch articles.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 411 P331 - A VERY RAPID EXTRACTION METHOD FOR DNA‑PROFILING OF BONE POWDER Francisca Elisabeth Duijs1, Titia Sijen1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

After a catastrophic disaster, bone material represents often the only resource for victim identification. Fast identification has high priority but DNA-profiling from bones is a time-consuming process. Generally, the best profiling results are obtained using demineralization protocols that aim to fully dissolve the bone matrix to free the DNA. These protocols often take 12 hours or more. Here, we describe the development of a rapid, semi-automated DNA extraction method based on partial demineralization. DNA is extracted from very finely grinded bone powder within the hour. Manual handling is minimal once the samples are loaded on the Maxwell FSC Promega instruments. When inhibitors are present in a bone sample, the short protocol may prevent them from being co-extracted resulting in better amplification of the DNA. We assessed how crucial the various steps in the protocol are and identified very fine granulation and full separation of bone powder remnants and DNA-containing supernatant as most important. This full separation was achieved through the use of Hamilton AutoLyse tubes prior to loading on the Maxwell FSC Promega instruments. The protocol will not extract all DNA present in a bone specimen, and is thus foremost suited for bones suffering limited levels of degradation. In those cases, the procedure offers a very rapid and semi-automated extraction protocol generating DNA profiles suited for DNA comparison. The success rates for casework samples will be discussed.

412 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P332 - ACHIEVING GREATER UNIFORMITY IN THRESHOLD DETERMINATIONS: RECOMMENDATIONS FOR STATISTICAL CONSIDERATIONS AND VALIDATION DESIGN Christian Westring1, Michaela Werner1, Phillip Danielson2

1 Purdue University Northwest, College of Engineering and Sciences, Hammond, Indiana, USA 2 University of Denver, Biological Sciences, Denver, Colorado, USA

Analytical and stochastic thresholds are used by many labs to discern allelic peaks from instrument noise and the potential for allele “drop-out”. It is recognized that the appropriate RFU value for these thresholds can maximize the informational value of a profile – especially in the context of mixtures or low-level contributors. Numerous statistical models for setting thresholds have been published and yet laboratories using the same instruments calibrated to the same manufacturer-specified standards can set substantially different thresholds (e.g., 40 to 250 RFU for the same chemistry and CE system). An important but oft-overlooked step in setting thresholds has been the consideration given to the distribution characteristics and thus the suitability of the underlying data sets used for a specific statistical method. This assessment is essential to ensuring the valid application of such common statistical tools as means, standard deviations and confidence intervals. In this study, we demonstrate the importance of recognizing and working with data distributions that are skewed or depart from normality. We demonstrate how failing to consider statistical and validation design issues can lead practitioners to a “false sense of confidence” with respect to allele detection and how this can lead to false inclusions/exclusions. Recommen- dations are provided for avoiding potential pitfalls by employing sound statistical principals. The critical need for “fit for purpose” thresholds is demonstrated as we assess the impact of assay sensitivity and detection enhancements when calculating stochastic thresholds. The methods summarized here will provide users with solutions for creating and properly applying statistics for establishing thresholds prior to implementation in casework.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 413 P333 - ADVANCED MITOCHONDRIAL CAPTURE ANALYSIS WITH MPS David Ballard1, Federica Giangasparo1, Anastasia Aliferi1, Laurence Devesse1, Skye Willis-Barrett1, Gabriella Mason-Buck1, Denise Syndercombe Court1

1 King’s College London, King’s Forensics, London, United Kingdom

The advent of massively parallel sequencing (MPS) technology has presented multiple and diverse opportunities to advance mitochondrial DNA (mtDNA) analysis, with the aim of improving assay sensitivity and robustness. One such method involves using mitochondrial capture probes – a technique that tolerates significant degradation to mtDNA and can generate full mitochondrial genome sequences. This approach foregoes the use of mtDNA specific PCR primers, but rather uses a shotgun sequencing approach coupled with a mtDNA hybridization capture assay. We present here mtDNA capture results from complex and badly damaged forensic samples including hair, bone and aged stain samples, all of which failed to produce autosomal DNA profiles. For comparison, capture sequences were assessed against results from traditional Sanger methods, as well as primer amplification based MPS methods as implemented in the Verogen (Beta-test) and Promega ‘mini-mito’ style kits (amplification of the control region via multiple overlapping small amplicons). The results from this comparison provide a framework to inform how mtDNA casework could be performed going forward.

414 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P334 - AN INNOVATIVE DNA EXTRACTION METHOD: WATER VERSUS COMMERCIAL BUFFERS Paula Mármol1, Beatriz Gómez1, Cláudia Gomes1,2, Carlos Baeza-Richer1,2, Ariana Hernández‑Cordero1, Marta Martín-Arrebola1, Rocío Rosell-Herrera1, Ana María López‑Parra1,2, Sara Palomo-Díez1,2, César López-Matayoshi1,2, Eduardo Arroyo-Pardo1,2

1 Facultad de Medicina Universidad Complutense de Madrid, Legal Medicine, Psychiatry and Pathology, Madrid, Spain 2 Instituto de Investigación Sanitaria del Hospital Clinico San Carlos IdISSC, Grupo de Ciencias Forenses: Genética y Toxicología forense, Madrid, Spain

The main objective of DNA extraction is to obtain good quality genetic material in order to carry out its amplification, and corresponding analysis. Most laboratories tend to resort to commercial extraction buffers, which allow a simple and rigorous DNA extraction, with limited handling of the sample (Gomes et al., 2017), but with high financial cost. As an alternative, we proposed to use water as reagent to extract DNA from blood spots, collected on Whatman® FTA® Cards (Sig- ma-Aldrich®). We propose to take advantage of hypotonic solutions, in order to obtain a better efficiency in the extraction of genetic material (DNA) On this sense, 233 extractions were performed with Whatman® FTA® Cards (Sigma-Aldrich®) stained with blood, by four distinct extraction methods. The four procedures are based on washing with different reagent volumes, and incubation with different temperatures and distinct shaking times. All techniques were performed with two types of water (sterile (water B|Braun, B. Braun Medical Inc) and distilled (Milli-Q, MilliporeSigma®)), as well as, with the commercial extraction kit (Prep-n-GoTM (ThermoFisher™ Scientific, Foster City, USA)). To evaluate and compare each reagent effectiveness, we amplified DNA extract with mitochondrial DNA Hypervariable regions I and II, and with nuclear DNA markers (autosomal STRs, Y-STRs and X-InDels). Our preliminary results suggest that distilled water allows an extraction as effective, or even better, as that obtained with the commercial buffer.These preliminary results offer the possibility for laboratories to choose an inexpensive protocol for DNA extraction, avoiding expensive commercial buffers. Further research is needed to evaluate the performance of each method in blood samples, but also in other biological samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 415 P335 - ANALYSIS OF 124 SNP LOCI INCLUDED IN HID AMPLISEQ IDENTITY PANEL IN A SMALL POPULATION OF RIO DE JANEIRO, BRAZIL Carolina Bottino1, Rosane Silva2, Rodrigo Soares Moura Neto3

1 IPPGF, Polícia Civil - RJ, Rio de Janeiro, Brazil 2 IBCCF, Universidade Federal do RIo de Janeiro UFRJ, Rio de Janeiro, Brazil 3 IB, Universidade Federal do Rio de Janeiro UFRJ, Rio de Janeiro, Brazil

Massively parallel sequencing platforms allow the simultaneous analysis of thousands to millions of DNA fragments, generating large amounts of data in one run and from very small amounts of material compared to traditional methods. Implementation of such analysis would mean a great saving of time and costs, as well as allowing reliable results to be obtained from small or extremely degraded samples. The aim of this work was to analyze 124 SNP loci (90 autosomal and 34 Y-SNP) included in HID Ampliseq Identity Panel in a small population of Rio de Janeiro, Brazil. Samples from 12 non-related individuals were amplified with HID Ampliseq Identity Panel and sequenced on the Ion Torrent PGM platform (Thermo Fisher Scientific); genotypes were generated with HID SNP Genotyper plugin and forensic parameters were calculated with PowerStats v.12. All samples were successfully genotyped and were used to calculate allele frequencies (0.958–0.042), homozygosity (0.833–0.083), heterozygosity (0.522–0.083), random match probability (RMP) (0.847–0.333), exclusion power (0.662–0.006) and polymorphic internal content (PIC) (0.375–0.077) for all 90 autosomal SNP loci. Among the 11 male samples analyzed, a prevalence of the haplogroup R1b of Y chromosome was observed, followed by the haplogroups E, Q and J. Such distribution reflects the results demonstrated in other studies for the population of Rio de Janeiro. All results together demonstrate the usefulness and applicability of SNP analysis on Ion Torrent PGM.

416 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P336 - ANALYSIS OF CASE WORK SAMPLE MIXTURES, LAST STEPS IN FORENSIC VALIDATION OF THE FORENSEQ™ DNA SIGNATURE PREP KIT Sallyann Harbison1, Ryan England2,3, Anne Harteveld4, Andrew Sarman2, Janet Stacey1

1 Institute of Environmental Science and Research Ltd, Forensic Biology, Auckland, New Zealand 2 Institute of Environmental Science and Research Ltd, Forensic Research Group, Auckland, New Zealand 3 School of Chemical Sciences, University of Auckland, Forensic Science Program, Auckland, New Zealand 4 University of Amsterdam, Master Forensic Science, Institute for Interdisciplinary Studies, Amsterdam, Netherlands

Massively parallel sequencing is fast emerging as an increasingly useful tool for forensic science. As the final steps of our validation of this technology we have carried out a DNA mixture study with up to 4 known contributors to test the performance of the system and it’s ability to generate informative profiles. We had three main aims. Firstly to compare the performance of MPS against our standard casework protocols using capillary electrophoresis. Secondly to determine the limit of detection of male DNA in a female background compared to Y STR DNA profiling and thirdly to assess the limitations of ancestry and phenotype prediction in mixed DNA samples. To do this we used the ForenSeq™ DNA Signature Prep Kit and a MiSeq FGx™ Sequencer. In this paper we describe the results of our studies and the strengths and weaknesses of a sequencing approach, addressing technical issues that require consideration when sequencing DNA from typical casework samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 417 P337 - ANALYSIS OF FORENSIC SAMPLES BY INVESTIGATOR ESSPLEX QS (QIAGEN) USING HALF PCR REACTION VOLUMES Anna Barbaro1, Patrizia Cormaci1, Angelo La Marca1

1 Studio Indagini Mediche E Forensi SIMEF, Forensic Genetics, Reggio Calabria, Italy

The Investigator ESSplex SE QS Kit (Qiagen) uses a fast-cycling technology for the simultaneous amplification, in just 60 minutes, of 16 STRs (including the 5 European Standard Set (ESS) markers and the SE33) plus the gender-specific locus Amelogenin. The kit amplifies also two Quality Sensors (QS1 and QS2) that acts as internal PCR controls to provide informations about PCR efficiency and the presence of PCR inhibitors. In this study we evaluated the performance of the InvestigatoESSplex QS using half reaction volume for the amplification of a wide range of casework samples bloodstains, vaginal swabs, semen stains, cigarette butts, saliva on bottles, contact traces, bones). Results showed that using half reaction volume (without altering ratio between components) is possible to obtain reliable DNA profiles, comparable to full-reaction data, also from difficult forensic samples: this confirms the Investigator ESSplex QS is a robust and sensitive multiplex.

418 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P338 - APPLICATION OF HRM-PCR (HIGH RESOLUTION MELTING PCR) FOR IDENTIFICATION OF FORENSICALLY IMPORTANT COLEOPTERA SPECIES Tadeusz Malewski1, Marta Los1, Magdalena Konarzewska2, Ireneusz Soltyszewski3

1 Museum and Institute of Zoology, Polish Academy of Sciences, Department of Molecular and Biometric Techniques, Warsaw, Poland 2 Medical University of Warsaw, Department Forensic Medicine, Warsaw, Poland 3 Warmia and Mazury University in Olsztyn, Department of Criminalistics and Forensic Medicine, Olsztyn, Poland

Precise estimation time of death is one of key task of forensic entomology. Application of Ca- lihoridae for post mortem interval estimation (PMI) is well suited for initial period of cadaver decomposition but limited to of their larval stage. Further stages of cadaver decomposition are associated with other entomofauna. Especially interesting is Coleofauna present at all stages of cadaver decomposition. Identification of Coleoptera species is however more difficult that Calliphoridae. Among different methods of species identification very promising is high-resolution melting PCR. Is allowes fast single-tube assignment of analysed sample to species based on amplicon melting profile. The object of this study were different specimens of Coleoptera collected at pig cadavers in Łomna (central Poland) during 2012 – 2014. Specimes were identified to species by experts of corresponding Coleoptera families. DNA from specimens were excracted by GenElute kit (Sig- ma-Aldrich). Primers to I subunit of cytochrome oxidase gene (COI) were designed by Prim- er3 software. Amplification were performed with RT HS-PCR Mix EvaGreen (A & A Biotechnol- ogy) at real time PCR thermocycler RotorGene 6000 (Qiagen). Data analysis was performed at RotorGene software. From 120 collected specimens belonging to 12 species HRM-PCR correctly identified 9 of them. Sequencing of amplicons from remaining three species showed differences in sequences what can indicate for presence of subspecies. The obtained test results indicate that t is more a precise tool rather than morphological research, which is traditionally used to determine the post mortem of the interim estimation

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 419 P339 - APPLICATION OF MHANALYSER SOFTWARE IN THE STUDY OF MICROHAPLOTYPES IN FORENSICS Zheng Li1, Yan Pu2, Jiawen Yang1, Weibo Liang3, Lin Zhang3, Feng Chen1, Peng Chen1

1 Nanjing medical university, Forensic medicine, Nanjing, China 2 Southeast university, School of medicine, Nanjing, China 3 Sichuan university, Forensic medicine, Chengdu, China

Microhaplotype is a short DNA sequence consisting of multiple SNPs, which is highly polymorphic and can be covered by one way reading length in massively parallel sequencing. Microhap- lotypes have been proved to have critical application value in many forensic aspects, such as individual identification, kinship analysis, mixture identification and ancestry inference. The file format obtained by using massively parallel sequencing is usually FASTQ, which could be analyzed by using FLfinder software 1. However, the analysis of microhaplotype data using FLfinder can only be carried out on a single individual. In addition, due to sequencing errors, multiple duplicate alleles would appear and complicate the analysis. Thus, dealing with multiple samples with multiple loci by using FLfinder is cumbersome and easy to make mistakes. On the basis of FLfinder software, we compile software MHanalyser, which can sort out the result of multiple samples and multiple loci simultaneously. The software MHanalyser is available at (http://forensic.njmu.edu.cn/html/8540761749.html). [1] Zhu J, Zhou N, Jiang Y, Wang L, He W, Peng D, Su Q, Mao J, Chen D, Liang W. FLfinder: A novel software for the microhaplotype marker[J]. Forensic Science International Genetics Supple- ment, 2015,5: e622-e624.

420 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P340 - APPLICATION OF NGM DETECT™ PCR AMPLIFICATION KIT IN CRITICAL FORENSIC EVIDENCE Gloria Brescia1, Venusia Cortellini1, Heitor Simoes Dutra Correa1, Laura Natalia Riccardi2, Nicoletta Cerri1, Andrea Verzeletti1

1 University of Brescia - Department of Medical and Surgical Specialties- Radiological Sciences and Public Health, Institute of Forensic Medicine, Brescia, Italy 2 Thermo Fisher Scientific, Thermo Fisher Scientific, Monza, Italy

The Applied Biosystems™ NGM Detect™ PCR Amplification Kit is a new Human Identification solution to increase sensitivity for casework samples with low DNA concentrations. The kit offers excellent sensitivity and provides an alternate marker configuration to the well-established NGM Select Kit format to maximize information recovery, even from degraded casework samples. The purpose of this study is to evaluate the performance of the NGM Detect™ kit compared to AmpFℓSTR® NGM Select™ and AmpFlSTR® Identifiler® Plus kits, using a variety of casework samples. The study is being carried out on samples obtained from forensic cases such as sexual violence, exhumations, paternity, homicides and suspicious deaths. The sample typologies refer to: buccal swabs, nail swabs, swabs deriving from sexual violence, human biological tissues and liquids, clothes and objects of common use. The preliminary results show that the combined use of NGM Detect and NGM Select kits provide maximum information in a dual amplification strategy, as the size of several markers are complementary. Furthermore, the NGM Detect and NGM Select kits appear more sensitive than the Identifiler Plus kit. In conclusion, together with the NGM Select™ kit, the NGM Detect™ kit offers the possibility of recovering a lot of genetic information from degraded samples, delivering the best probability of identity value compared to other ESS-focused solutions in the market today.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 421 P341 - APPLICATION OF PARTIAL STR PROFILES GOTTEN BY ARTIFICIAL DNA MIXTURE DECONVOLUTION Jing Chen1, Zheng Tu1, Wanshui Li1, Chong Wang1

1 Institute of Forensic Science- Ministry of Public Security- P.R.China, Forensic Genetics, Beijing, China

DNA mixture is common to be obtained in forensic DNA test, as the complexity of crime scenes and the increasing sensitivity of DNA testing kits. How to make better use of DNA mixture has always been a hot spot for DNA experts. Recently, some professional software has been widely and deeply applied in DNA mixture deconvolution, and achieved great success. The software lists multiple STR profiles, according to the principle of permutation, and performs pendulum query for the technicians to use. However, it is another good way which combines the powerful DNA database and partial STR profiles gotten by artificial DNA mixture deconvolution, to make the DNA mixture become useful. This method is especially suitable for cold cases or the situations of losing original analysis data. When facing difficult 2 (existing random amplification) or 3 contributors’ profiles, first of all, try to list the partial profiles of major contributor(s) or/and minor contributor(s) from the profile, according to the peak height and peak area of multiple parallel amplification results. Secondly, the partial profiles should have 7 loci at least. However, the loci can be grouped according to the principle of permutation, which will constitute different profiles. Then, type the target partial profiles into DNA database to get hit with a properly magnified tolerance ( 2 pairs). Finally, check whether the other sites are consistent after the database results, and take more background information into consideration before the database results are determined≦ to be reliable.

422 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P342 - APPLICATION OF THE GNANO 31-PLEX ANCESTRY PREDICTION ASSAY IN AN AUSTRALIAN CONTEXT Catherine Hopkins1, Duncan Taylor1,2, Kelly Hill2, Julianne Henry1,2

1 Flinders University of South Australia, College of Science and Engineering, Adelaide, Australia 2 Forensic Science SA, Biology, Adelaide, Australia

The Global AIMS Nano (GNANO) 31-Plex SNaPshot assay can successfully differentiate five main continental populations; African, European, East Asian, Oceanian and Native American. We have recently verified GNANO for implementation into DNA casework at Forensic Science SA as a reliable and cost-effective alternative to an MPS-based assay. The South Australian population is mainly composed of Europeans (95 %), East Asians (3 %) and Australian Aborigines (2 %). Whilst some remote tribal Aboriginal populations still exist, the majority of contemporary Aborigines are impacted by European admixture. Whilst the reliability of the GNANO assay to infer European and East Asian ancestry has already been established, its performance for Australian Aborigines was untested. GNANO profiles were generated for 97 self-declared Australian Aborigines and analysed using Snipper and STRUCTURE. Aboriginal individuals presented as a continuous genetic cline between the Oceanic and European populations, depending on the level of European admixture. Some divergence in genotypes was observed when individuals from remote (non-admixed) tribal regions were compared to individuals from more urbanised (admixed) regions. When added as a separate reference population, 74 % of self-declared Aboriginal individuals were inferred as Aboriginal. Whilst the ability of GNANO to predict Aboriginality at a genetic level was reasonable, we do not feel that the assignment of an individual as either ‘Australian Aboriginal’ or not is an appropriate reporting framework. This is because it may prejudice investigators towards a particular phenotype and it does not account for a person’s social affiliation. Rather, we consider reporting a breakdown of the ancestral makeup of an individual to be more appropriate.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 423 P343 - APPLIED BIOSYSTEMS™ VERIFILER™ PLUS KIT WITH INTERNAL QUALITY CONTROL SYSTEM PROVIDES CONFIDENT ANSWERS IN CHALLENGING FORENSIC SAMPLES Angela Lackey1, Jianye Ge1, Chang Zhong1, Robert Green1, Wilma Norona1, Julio Mulero1

1 Thermo Fisher Scientific, Human Identification, South San Francisco, USA

The Applied Biosystems™ VeriFiler™ Plus PCR Amplification Kit has been developed to meet challenging forensic casework needs globally. It is a companion kit to the Applied Biosystems™ VeriFiler™ Express PCR Amplification Kit for databasing. Powered by a six-dye short tandem repeat (STR) chemistry and an improved master mix formulation, this kit is specifically designed for challenging sample types—touch, degraded, or inhibited samples with improved sensitivity and enhanced robustness against inhibition. With a total of 25 loci- 23 autosomal STRs, including Penta D and Penta E, and 2 gender discrimination markers, the VeriFiler Plus kit also includes an innovative internal quality control (IQC) system. The IQC system contained in the Verifiler Plus kit consists of two synthetic sequences with specific primers for each of the targets. This system provides positive confirmation that all assay components are functioning as expected. This system is particularly useful for confirming the validity of negative results and distinguishing samples that are degraded from those that contain PCR inhibitors. When the IQC system indicates degraded DNA, forensic analysts may reamplify a forensic sample with a higher amount of input DNA or choose a complementary short tandem repeat (STR) amplification kit that has an alternative marker set configuration to maximize information recovery. If the IQC system indicates presence of inhibitors, the analyst may opt for an additional purification step or a dilution of the original sample before repeating sample amplification.

424 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P344 - ASSESMENT AND PREVENTION OF FORENSIC DNA CONTAMINATION IN DNA PROFILING FROM LATENT FINGERPRINT Ketsaraporn Nontiapirom1, Wanasphon Bunakkharasawat1, Punchapat Sojikul2, Nathinee Panvisavas3

1 Faculty of Science- Mahidol University, Forensic Science Unit, Bangkok, Thailand 2 Faculty of Science, Mahidol University, Biotechnology, Bangkok, Thailand 3 Faculty of Science, Mahidol University, Forensic Science Unit / Department of Plant Science, Bangkok, Thailand

In this study, fingerprint brush contamination and a simple fingerprint brush cleaning procedure was assessed. A total of 10 new camel-hair fingerprint brushes were used. Prior to the experiments, 10 hairs were collected from each brush to check their background DNA profile. Results showed that 1 allele was called once from 5 brushes. No DNA profile was generated from the black dusting powder. Then, the fingerprint brushes were used for dusting either fresh saliva or saliva stain prior to dusting 4 latent fingerprints deposited on the glass surface. Ten hairs were cut from each fingerprint brush and 4 latent fingerprints were collected by double swab technique for DNA analysis. Consensus DNA profiles of samples were generated from 3 replications of DNA profiling. Mixed-DNA profiles were obtained from the latent fingerprint swabs. More than 2 alleles were observed, and matched alleles from the saliva and fingerprint donors. DNA profiles generated from the brush hairs had no complete loci. The called alleles matched the saliva donor and some were shared alleles of the 2 contributors. Amount of DNA deposited on fresh-saliva contaminated brushes were higher than that those contaminated with saliva stain. Next, fingerprint brushes were cleaned with dish-washing liquid and rinsed with sterile-water. DNA profiling of 5 cleaned-brushes showed that no consensus DNA profile was obtained. Although few alleles were called in each replication of the DNA profile, they were not reproducible. The persistence of biological material on fingerprint brush hairs was demonstrated, thus being a potential source of contamination by indirect material transfer. Cleaning fingerprint brush after direct exposure to biological fluid and stains could help to prevent evidence cross-contamination.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 425 P345 - ASSESSING DNA RECOVERY FROM HIGHLY DEGRADED SKELETAL REMAINS BY USING SILICA-BASED EXTRACTION METHODS Diana Vinueza Espinosa1, Cristina Santos1, Cristina Martinez-Labarga2, Assumpció Malgosa1

1 Autonomous University of Barcelona, Department of Animal Biology, Vegetal Biology and Ecology/ Biology Anthropology Research Group, Barcelona, Spain 2 Tor Vergata University- Italy, Department of Biology, Centre of Molecular Anthropology for Ancient DNA Studies, Roma, Italy

The human DNA recovery from highly degraded skeletal remains is the most limiting factor in forensic genetics research. In cases of missing persons and mass disasters, the bones may be the only suitable material available to identify individuals or their family relationship. Nonethe- less, the use of relatively specialized techniques is necessary for the obtaining of any genetic information, particularly when the remains have been exposed to extreme environmental factors and DNA is degraded resulting in low concentration, fragmentation and molecular damage. The aim of this study is to assess the DNA recovery of different extraction methods and the amplification efficiency of PCR-amplifiable nuclear DNA (autosomal STRs by Global Filer) and mitochondrial DNA (mtDNA). Samples from five type of bones from five individuals (5–12th centuries AD) were selected. The DNA extraction was processed individually by using four extraction methods, (N:100 DNA extracts), three based on DNA adsorption to silicon dioxide (silica) particles, Non-Columns Silica (NCSi), Silica-HE Spin Columns (SiHEC), Silica-XS Spin Columns (SiX- SC) and the another one is the traditional organic extraction method (P-Chl). The bones selected were petrous, tooth pulp cavity, tooth cementum, postcranial bones (radial, ulna, metacarpal or phalange) and rib. The results showed that the NCSi and SiHEC extraction methods, petrous bones and pulp cavity allowed to recover the highest amount of PCR-amplifiable human mtD- NA. The autosomal STRs profiles from petrous presented >70 % reportable alleles and peak heights between 200–500 RFUs. The lower amount of DNA was showed from rib. The outcomes variability was also dependent of the individuals, being this a valuable factor when we evaluated the efficiency of each protocol.

426 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P346 - ASSESSING MIXTURE ANALYSIS UTILIZING SNP PROBE CAPTURE ENRICHMENT AND MASSIVELY PARALLEL SEQUENCING Jessica Lim1,2, Shelly Shih2, Henry Erlich2, Cassandra Calloway1,2

1 Forensic Science Graduate Group, University of California - Davis, Davis, California, USA 2 Children’s Hospital Oakland Research Institute, University of California San Francisco Benioff Children’s Hospital, Oakland- California, USA

DNA evidence found at crime scenes and mass disaster events can be highly degraded and mixed in nature, creating problems for STR analysis by CE. Highly degraded samples typically fail STR analysis due to the absence of both intact primer binding sites in the template DNA, resulting in PCR amplification failure. In addition, current methods of STR analysis by CE are unable to detect minor contributors present at less than 10 % in mixture evidence. Single nucleotide polymorphisms (SNPs) offer an alternative method in the analysis of highly degraded samples due to their small genetic footprint. Multi-allelic markers and haplotypic Y-SNPs can help in the resolution of mixtures compared to bi-allelic SNPs alone. Probe capture enrichment coupled with next-generation sequencing (NGS) technology alleviates the need for intact primer binding sites by using a highly redundant tiling strategy to capture targeted regions of interest, enabling the analysis of highly degraded mixtures which would typically fail STR analysis due to the short DNA fragment size. DNA mixtures can be clonally sequenced, allowing for bioinformatic read counting to estimate minor contributions. We applied two versions of a SNP probe capture enrichment panel targeting 426 (v1.0) and 436 (v2.0) SNPs to contrived mock-degraded and control mixtures. Varying DNA amounts ranging from 25 ng – 1 ng were assessed with minor contributions ranging from 2.5 % – 50 %. The detection of a minor component was achieved at a minor contribution of 2.5 %. Minor contributions were accurately and precisely estimated in the contrived mixtures tested. We show proof-of-concept of the application of SNP probe capture enrichment in the analysis of highly degraded mixtures where conventional STR analysis may fail.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 427 P347 - ASSESSMENT OF DNA QUANTITY AND QUALITY IN A WIDE RANGE OF FORENSIC SAMPLES USING THE INVESTIGATOR QUANTIPLEX PRO RGQ KIT (QIAGEN) Anna Barbaro1, Patrizia Cormaci1, Angelo La Marca1

1 Studio Indagini Mediche E Forensi SIMEF, Forensic Genetics, Reggio Calabria, Italy

The Investigator Quantiplex ProRGQ kit (Qiagen) is a new kit that uses a fast-cycling technology for PCR real-time quantification, in around 1 hour, of total human DNA and male DNA. The assay provides, simultaneously, reliable informations about DNA inhibitors persistence, degradation status and possible mixture condition. In the present study, we evaluated the performance of the Investigator Quantiplex Pro RGQ Kit on a wide range of forensic samples (biological fluids, blood stains, semen stains, vaginal swabs, oral swabs, cigarette butts, saliva traces on objects, contact traces, bones, teeth, cadaveric tissues, carbonized tissue, paraffin-embedded tissues, formalin fixed tissues) using the Real Time instrument Rotor-Gene Q (Qiagen). Data obtained have been analyzed by the software Q-Rex (Qiagen) together with the Quant Assay Data Handling Tool: quantification results, Inhibition Index (IC), Degradation Index (DI), Mixture Index (MI) have been evaluated. As expected, differences in DNA quantity and quality were found, due to the nature of substrates and consequently different ability to preserve and release biological samples. In all cases, we observed a full concordance between quantification data and the quality of STRs profiles produced using the Investigator ESSplex SE QS kit (Qiagen). This study confirmed the Investigator Quantiplex Pro RGQ Kit provides accurate and precise results with a high sensitivity of both quantitative and qualitative assay.

428 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P348 - ASSESSMENT OF THE DNA REPAIR FOR RESTORATION OF STR PROFILES FROM DAMAGED MIXTURES WITH VARIOUS RATIOS Eun Hye Kim1, Jung Yoon Lee1, Sang Hyun An1, Saimi Jung1, Mu-Yeong Lee1, Dong-ho Choi1, Kim Jong Jin1

1 National Forensic Service Seoul Institute, DNA analysis Division, Seoul, Republic of Korea

The deconvolution of the DNA mixtures from various types of the evidence is important to distinguish true alleles from artifacts and help mixture interpretation. However, the mixture from forensics sometimes exposes them to environmentally severe conditions and various damage- able factors. The analysis of damaged mixtures will affect incorrect STR typing caused by stochastic effects and loss of larger loci due to degradation. This study demonstrated that the Pre- CR™ repair enzyme was applied to the UV-exposed mixtures with various mixed ratios. Mixtures of two control DNAs were prepared in mixture ratios of 1:1, 1:3, 1:6, 1:9, 1:19, 1:29 and 1:49 (male: female). Assessment of the restored DNA profiles was analyzed to detect DNA degradation index (DI) using Quantifiler® Trio DNA quantification kit and evaluate of the repaired STR profiles using PowerPlex® Fusion PCR kit and Globalfiler® kit. STR profiles were analyzed and results for both PCR methods were compared. Consequently, we will illustrate the results of the effect of DNA repair treatment on various mixture ratios. This result will be used to suggest the potential usability of the DNA repair system for restoration of STR typing in actual forensic caseworks. Additionally, further research of the DNA repair for restoration of STR profiles in real caseworks is ongoing to evaluate the potential for application to forensic DNA analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 429 P349 - AUTOMATED ESTIMATION OF THE NUMBER OF CONTRIBUTORS IN AUTOSOMAL STR PROFILES Jennifer van der Linden1, Jerry Hoogenboom1, Corina Benschop1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

The number of contributors (NOC) to (complex) autosomal STR profiles cannot be determined with absolute certainty due to complicating factors such as allele sharing and drop-out. The accuracy of NOC estimates can be improved by increasing the number of (highly polymorphic) markers, the use of massively parallel sequencing instead of capillary electrophoresis, and/or using more information than only the allele counts. In this study, we focussed on methods to automatically estimate the NOC and used -amongst others- a machine learning approach in order to make maximum use of the profile information. To this aim, a set of 590 PowerPlex Fusion 6C profiles with one up to five contributors was used. This set varied for the template amount of DNA, mixture proportion, levels of allele sharing, drop-out and degradation. The dataset was split into a training, test and validation set. The training set contains labels with the known NOC and was used to train and optimize nine different algorithms with selection of profile characteristics. Per profile, over 250 characteristics, denoted ‘features’, were calculated. These features are based on allele counts, peak heights and allele frequencies. The features that are most related to the NOC were selected based on partial correlation using the training set. Next, the performance of each model (=combination of features plus algorithm) was examined using the test set containing unlabelled data. The model that resulted in best training and test accuracies was selected for further validation. Results show improved accuracies compared to conventional allele counting approaches. The method is very fast and regarded useful for application in casework. Therefore, this method will be integrated in our DNA eXpert System, denoted DNAxs.

430 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P350 - BACKGROUND NOISE ASSESSMENT OF ILLUMINA MITOGENOME DATA FOR HETEROPLASMY DETECTION Kimberly Andreaggi1,2, Charla Marshall1,2

1 SNA International, n/a, Alexandria, USA 2 Armed Forces Medical Examiner System, Armed Forces DNA Identification Laboratory, Dover, USA

Massively parallel sequencing (MPS) was first introduced into forensic laboratories for mitochondrial DNA (mtDNA) analysis. The high-throughput capabilities of MPS have allowed the sequencing of the entire mtDNA genome (mitogenome), increasing the discrimination power of the locus. The quantitative nature of MPS data offers many benefits to mitogenome analysis; yet this requires the determination of thresholds for variant calling such as minimum coverage and minimum variant frequency. This study assessed background noise observed in the mitogenome data of diverse haplotypes in order to characterize the limit of detection of Illumina sequencing chemistry. Libraries were generated from overlapping long-range PCR products targeting the entire mitogenome, and sequenced on Illumina MiSeq FGx Systems. Sequencing was performed with V2 and V3 MiSeq chemistries, paired- and single-end reads, increased levels of multiplexing (12, 48 and 96 samples), and three different MiSeq systems. Overall, the average background noise observed was 0.15 % (± 0.25 %) regardless of the multiplexing level, MiSeq chemistry, MiSeq instrument, or sequencing method (paired- vs single-end). Based on the background assessment, the minimum variant frequency that should be applied for mtDNA heteroplasmy detection is between 0.9 % (average + 3σ) and 2.65 % (average + 10σ). However, different sample processing methods (e.g., hybridization capture for enrichment, sequencing on the Illumina NextSeq 550 System) and analysis pipelines may require a minimum variant detection threshold >3 % due to elevated background noise. Disclaimer: The opinions and assertions presented hereafter are the private views of the authors and should not be construed as official or as reflecting the views of the United States government.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 431 P351 - BIOLOGICAL STAIN COLLECTION – ABSORBING PAPER IS SUPERIOR TO COTTON SWABS Kirstin Janssen1, Marthe Aune1, Marita Olsen1, Gunn-Hege Olsen1, Thomas Berg1

1 UiT The Arctic University of Norway, Centre of Forensic Genetics, Institute of Medical Biology, Faculty of Health Sciences, Tromsoe, Norway

Biological evidence at crime scenes often contains very small amounts of DNA. Therefore, it is important to use the most effective sampling devices and procedures for stain collection. Currently, cotton swabs moistened with water are widely used, also in our laboratory. However, several studies have shown that other methods may be more efficient. In this study, we compared the DNA sampling efficiency of cotton swabs (Puritan) and pieces of absorbing paper (Kimtech) moistened with two liquids, water and ethanol. An initial experiment with blood stains deposited on glass slides showed that DNA yields were highest for samples collected with absorbing paper and ethanol. For cotton swabs, using ethanol instead of water did not increase the DNA yield. To reflect casework conditions, we tested cotton swabs with water versus absorbing paper with ethanol on a range of objects and clothing that had been in use and for which it was possible to define two equivalent areas for stain collection. Objects were divided into four surface classes: leather, plastic, natural and synthetic fabrics. We found that DNA yields were higher when using absorbing paper and ethanol than with cotton swabs and water. These findings were significant for all surface classes except synthetic fabrics for which there was a trend in the same direction though. These results suggest that pieces of absorbing paper moistened with ethanol can improve the efficiency of stain collection, especially when stains are expected to contain low amounts of DNA. However, user-friendliness could still be improved and contamination risk reduced if an easy-to-handle collection device based on absorbing paper was developed.

432 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P352 - BRINGING FORENSIC Y-CHROMOSOME HAPLOGROUPING TO THE NEXT RESOLUTION LEVEL BY USING TARGETED MASSIVELY PARALLEL SEQUENCING Arwin Ralf1, Mannis van Oven1, Diego Montiel González1, Peter de Knijff2, Kees van der Beek3, Sharon Wootton4, Robert Lagacé4, Manfred Kayser1

1 Erasmus MC University Medical Center Rotterdam, Department of Genetic Identification, Rotterdam, Netherlands 2 Leiden University Medical Center, Department of Human Genetics, Leiden, Netherlands 3 Netherland Forensic Institute, Custodian DNA database, The Hague, Netherlands 4 Thermo Fisher Scientific, Human Identification Group, South San Fransisco, USA

Y-chromosomal single nucleotide polymorphisms (Y-SNPs) are useful DNA markers in forensic genetics as they allow for paternal lineage identification and for paternal bio-geographic ancestry inference. Previously, forensic Y-SNP analysis was mostly performed via single base extension (SBE) using SNaPshot chemistry. Although, this technology is highly sensitive, making it suitable to forensic DNA-analysis, it comes with strong limitations in the number of SNPs that can be analyzed simultaneously. Consequently, most previous Y-SNP genotyping tools only provided limited answers for paternal lineage and ancestry inference. Targeted massively parallel sequencing (MPS) allows for the parallel analysis of hundreds of SNPs as we previously demonstrated with a Y-SNP MPS-tool. In its recently revised version, this MPS tool targets 859 Y-SNPs and 640 Y haplogroups, comes with a revised software for data analysis including Y haplogroup calling, and is able to cope with low quality and low quantity DNA as demonstrated in the forensic developmental validation study performed. The AmpliSeq primer pool is made commercially available as custom panel under the name Ion AmpliSeq HID Research Panel V1 (Thermo Fisher Scientific). Through this poster, we describe key elements of this methodology. Moreover, due to the lack of population data from many of the Y-SNPs our tool targets, we use this poster to call-up for a multi-center study seeking for worldwide participants to apply our tool in their local population samples. The global haplogroup frequency data generated through this future collaboration will be made publically available to provide a valuable resource benefitting the field of forensic genetics, and additional fields such as genealogy and human population history.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 433 P353 - CAPTURING SPERMATOZOA FOR STR ANALYSIS OF SEXUAL ASSAULT CASES USING ANTI-SPERM ANTIBODIES Dina Alsalafi1, William Goodwin2

1 Department of Forensic Science, Sharjah Police General HQ, Sharjah, United Arab Emirates 2 School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, United Kingdom

DNA isolation in sexual assault cases is complicated by the presence of large numbers of female epithelial cells, which are often in vast excess when compared to spermatozoa. The two-step differential extraction has become the standard method for isolating the spermatozoa; however, new techniques in forensic science allow the use of precision techniques for capturing spermatozoa. In an attempt to refine the process we have used an immuno-magnetic bead-based technique for sperm cell separation. We identified two antibodies that were specific to spermatozoa: SP17 polyclonal antibody and SP10 Intra Acrosomal Protein monoclonal antibody. These were conjugated to Dynabeads® M-450 Epoxy beads. We used these antibody conjugates to isolate the spermatozoa in samples that exhibited the characteristic of sexual assault samples. Microscopy showed the successful separation of spermatozoa in the samples with sperm concentration 104/ml and 103/ml and STR analysis produced full male profiles, similar to the result of the samples extracted using the two-step differential method. Mixed profiles were seen for the samples with 102/ml sperms and incomplete male profiles with female STR peaks as major contributor when the density of the samples were 101/ml in comparison to the established two-step differential method. As a result, our finding suggested the possibility to isolate the spermatozoa of sexual assault samples using magnetic beads coupled to antibody against sperm specific proteins and it could be available for application in routine work as an alternative to conventional methods.

434 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P354 - CHARACTERISATION OF THE IMPACT OF INHIBITION ON STR PROFILES: CAUSES, MECHANISMS AND CONSEQUENCES David Moore1

1 Eurofins Forensic Services, DNA Research and Development, Teddington, United Kingdom

Inhibition of the polymerase chain reaction can jeopardise the success of STR genotyping. A number of studies exist that have looked at the impact of inhibitors on STR profiling; these generally look at the broad impact of inhibition, such as the number of peaks observed, average peak height and profile success. This study investigates inhibition in more detail by comparing patterns of inhibition with known characteristics of an STR kit. By looking at the balance of the loci within STR profiles under different inhibitory conditions and assessing the impact of altering PCR conditions such as annealing temperature on locus performance, hypotheses of inhibitory mechanisms can be formed. The data show that a number of inhibitory mechanisms could be identified. Some of these appeared to be associated with primer annealing, where loci with primers with lower annealing temperatures were observed to be inhibited more than loci with primers with higher annealing temperatures. Other inhibitors appeared to impact on the processivity of the polymerase enzyme affecting high molecular weight loci more than low molecular weight loci; this produces an effect similar to degradation with the classic “ski-slope” pattern. These data can be used to inform what inhibitors may be present within a sample for troubleshooting purposes. Addition- ally, these data provide information that can be used to improve primer design for improved inhibitor tolerance.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 435 P355 - CHARACTERIZATION OF A 41-PLEX PCR AMPLIFICATION ASSAY FOR MALE-SPECIFIC DATABASING APPLICATIONS Siddhita Gopinath1, Jianye Ge1, Julio Mulero1, Andrea Carbonaro1, Marc Short1, Matthew Ludeman1, Angela Lackey1

1 Thermo Fisher Scientific, Human Identification, South San Francisco, USA

DNA databases are indispensable tools in forensics to help solve crimes by matching autosomal STR profiles obtained from crime scene samples with known crime offenders. In more recent years the forensic community has been debating the inclusion of Y-STR markers to existing databases to help determine or exclude relationships, identify missing persons, infer ancestry and interpret mixture. We developed a 41-plex that simultaneously amplifies the 27 Y-STR markers included in the Applied Biosystems Yfiler™ Plus PCR Amplification Kit plus 11 new Y-STRs (DYS549, DYS645, DYS557, DYS593, DYS522, DYS444, DYS596, DYS643, DYS447 and DYS527a/b) and 3 Y-indels, which together can provide extremely high discriminating power. This multiplex is designed to process single-source reference samples using direct PCR amplification from blood samples on paper substrates without the need for sample purification. This 41-plex was built in a 6-dye multiplex format with PCR products ranging from 68–570 base pairs and it is compatible with detections on the 3130xl, 3500xL and 3730xl instrument platforms. Particularly, the Y-indels can be used to quickly exclude male lineages, as the mutation rates of the Y-indels are significantly lower as compared to STR’s (i.e., ~10-9 vs 10-3 per locus per generation). This study shows the feasibility of amplifying 41-markers simultaneously and describes the optimization of the multiplex assay to deliver high first pass success rate and assay performance when amplifying blood samples on paper substrates. In addition, the haplotype diversity and discriminatory capacity calculations with the expanded multiplex will be presented. For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

436 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P356 - CHARACTERIZING STUTTERS IN FORENSIC STRs WITH MASSIVELY PARALLEL SEQUENCING Ran Li1, Riga Wu1, Haixia Li1, Xuefeng Shen1, Yinming Zhang1, Hongyu Sun1

1 Sun Yat-sen University, Faculty of Forensic Medicine, Zhongshan School of Medicine, Guangzhou, China

Despite of the improvement in characterizing stutter of short tandem repeat (STR), the relationship between stutters and the relationship between motifs are not well understood yet. In the present study, 750 samples were sequenced to characterize the stutters of 58 STRs using ForenSeq™ DNA Signature Prep Kit. With a sequence simplification procedure, alleles and stutters were identified without ambiguity. After screening, 26921 alleles were included, resulting in over 50 million reads, 8.71 % of which were stutters. N-1 stutters accounted for the largest fraction of these stutters with a value of 83.36 %. N-4, N-3, N-2, N0, N+1 and N+2 stutters consisted of 0.18 %, 0.80 %, 6.43 %, 2.99 %, 5.93 % and 0.28 %, respectively. Stutters correlated best with corresponding one unit longer stutters (or parental allele) for backward stutters while N+1 stutter correlated best with N-1 stutter rather than the expected parental allele. Besides, we found that the motifs of D21S11 stuttered differently. Interrupted motifs stuttered independently while they did not for continuous motifs. In conclusion, with MPS technology, sequence variations within alleles and stutters can be identified, which is helpful in determining the origin of stutters and improving the resolution for mixture analysis. One repeat unit longer stutters are better predictors for the backward. Additionally, a co-stuttering pattern exists between the two motifs adjacent to each other while interrupted motifs stutter independently.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 437 P357 - CHIMERISM ANALYSIS USING NEXT GENERATION SEQUENCING Barbara Minuti1, Anna Lari1, Sara Iozzi1, Simona Palchetti1, Beatrice Boschi1, Francesca Gerundino1, Ugo Ricci2, Elisabetta Pelo1

1 Azienda Ospedaliero Universitaria Careggi, SOD Diagnostica Genetica, Florence, Italy 2 AOU Careggi SOD Diagnostica Genetica, Forensic Genetics Unit, Florence, Italy

Hematopoietic stem cell transplantation (HSCT) is the predominant curative treatment for many malignant and non-malignant haematological diseases. In order to evaluate the level of donor engraftment, mixed chimerism levels must be carefully monitored after transplantation. Short-tandem repeat (STR) genotyping is widely used to determine the proportions of donor and recipient cells after HSCT. In this study, Devyser Chimerism NGS kit in combination with a MiSeq System was introduced in our laboratory for monitoring HSCT. This system is a complete workflow solution for labs, combining a reliable testing process with a designed for-purpose analytical software. Streamlined, simple and robust NGS workflow uses just one multiplex PCR reaction per patient sample. Minimal hands-on time reduces assay complexity and risk of sample contamination and mix-up. User-friendly, designed-for-purpose software perfectly complements testing kit with an automatic detection of informative markers. Up to 24 informative markers in a recipient/donor pair distributed through the human genome have strong discriminative power with low bias from ethnic parameters. These IND/DEL genetic markers with population independent discriminative power are distributed across 17 chromosomes and were further selected to allow sensitive detection combined with accurate and precise quantification of mixed chimerism. These polymorphisms have been used in the fields of forensic investigations owing to the advantages of their low mutation rates, widespread distributions in the human genome and small amplicon sizes. Thus, and evaluation of Devyser Chimerism NGS kit for forensic individual identification was also tested.

438 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P358 - COMPARATIVE ANALYSIS OF DIFFERENT DNA RECOVERY METHODS FROM TOUCH DNA DEPOSITED ON PLASTIC BAGS AND ALUMINIUM FOIL Anja Radanovic1, Miljana Kecmanovic1, Vanja Tanasic1, Milica Mihajlovic1, Verica Radojicic1, Fedja Puac1, Milica Keckarevic Markovic1, Dusan Keckarevic1

1 University of Belgrade, Faculty of Biology, Department for Biochemistry and Molecular Biology, Belgrade, Serbia

Throughout the last few decades, much interest has been given to touch DNA samples. DNA from presumably touched or handled items is often degraded, but today, due to higher sensitivity of commercially available kits and technological improvements, DNA profile could be obtained from only a few cells. Hence, touch DNA samples are not challenged but are considered as standard samples in forensic casework nowadays and their use in courtrooms of forensic DNA typing increased. Factors impacting transfer, persistence and recovery of DNA, as well as quality of DNA profiles are still a subject of numerous studies. The aim of our research was to investigate touch DNA samples in form of fingerprints from three volunteers deposited on two different items. We studied shedding status of volunteers, type of material on which DNA was deposited, two methods of DNA isolation and hand dominance as factors that could potentially influence quantity of DNA recovered and quality of DNA profiles. We chose plastic bags and aluminium foil as DNA deposition materials because we wanted to imitate realistic conditions in which drugs were often packed. Our results demonstrated that DNA extraction performed on the same day as DNA deposition yields much better results, with higher DNA concentration and higher quality of DNA profiles, compared to the extraction that requires overnight incubation. We also noticed substantial differences in DNA concentration and overall quality of DNA profiles in a female volunteer compared to males. Our research has also shown that two types of tested materials and hand dominance do not affect quantity of recovered DNA or quality of obtained DNA profiles in tested conditions.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 439 P359 - COMPARATIVE STUDY ON THE EFFECTS OF REDUCED PCR REACTION VOLUMES OF THE FORENSEQ™ DNA SIGNATURE PREP KIT Stefania Turrina1, Domenico De Leo1

1 University of Verona, Department of Diagnostics and Public Health, Verona, Italy

The introduction of the Massively Parallel Sequencing (MPS) technology in forensic genetics field has been evaluated positively by scientific community, especially because it complements the weak points of capillary electrophoresis (CE) in genetic markers analysis. However, one of the main obstacles to its practical application seem to be the costs of the MPS library kits used. As reported in several published studies concerning the typing of STRs by CE, the reduction of the PCR reaction volume to half or one-third of its original volume, makes it possible to limit the kit expenses without affecting the ability to obtain a DNA profile and the quality of this one. Therefore, we evaluated whether the volume reduction of PCRs included in the preparation of the MPS library has an adverse effects on the resulting DNA profiles. This approach has been assessed using the ForenSeq DNA Signature Prep Kit. In the first step of library preparation, in which genomic DNA is amplified and tagged using a primer mix (in this study only DNA Primer Mix A was considered), the PCR was optimized on 1 µl of DNA at concentration of 1ng/µl instead of 5 µl recommended by the manufacturer and all the other steps of library preparation were optimized on this one. DNA sequence profiles obtained with this approach were compared to conventional library preparation and no difference in depth of coverage and allele coverage ratio were detected.

440 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P360 - COMPARING FORENSEQ AND GENOME-WIDE SNP DATA FOR KINSHIP DEDUCTION Margherita Colucci1, Burkhard Rolf2, Martin Blythe3, Nuala Sheehan4, Mark A. Jobling1

1 University of Leicester, Department of Genetics and Genome Biology, Leicester, United Kingdom 2 Eurofins Medigenomix Forensik, GmbH, Ebersberg, Germany 3 DNA Worldwide Group, Living DNA, Frome, United Kingdom 4 University of Leicester, Department of Health Science, Leicester, United Kingdom

Kinship estimation has application in forensic casework where the deduction of relationships between individuals can aid in the identification of suspects. Multilocus genotype data derived from genome-wide SNP chips provide a high degree of power, but are not generally practical for the sample types encountered in forensic casework. The ForenSeq DNA Signature Kit can analyse up to 230 markers, including autosomal SNPs, and autosomal, X-chromosomal and Y-chromosomal STRs, via PCR followed by massively-parallel sequencing (MPS). Here, we compare the performance of SNP-chip and ForenSeq DNA analysis for a set of 72 individuals belonging to 8 pedigrees of German ancestry. Genome-wide data from the Illumina HumanOmniExpressEx- ome-8 v1.2 chip (964,193 SNPs, including autosomal, 22,927 X, 1506 Y and 218 mtDNA SNPs) were obtained, and the performance of different methods (PLINK, PRIMUS and GENESIS) in estimating relationships from autosomal data evaluated. In this non-admixed sample set the methods perform comparably. We also explored the additional power from adding information post facto on X, Y and mtDNA markers. We present a comparative analysis based on ForenSeq data on the same set of samples, using both conventional autosomal STR-based methods and a com- binatorial approach that accounts for STR mutation within a framework of SNP-based inference, and also incorporates Y- and X-STR data.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 441 P361 - COMPARISON OF SILICA FIBRES COATED WITH CHITOSAN FOR THE EFFECTIVE CAPTURE AND RELEASE OF DNA Jarrad Rennie1, Piyamas Kanokwongnuwut1, Adrian Linacre1, Paul Kirkbride1

1 Flinders University, College of Science and Engineering, Adelaide, Australia

We report on the collection of epithelial cells on various surfaces using a specifically designed forensic swab which both efficiently recovers and releases touch DNA samples. Here we compare the efficiency of a new swab with those available commercially in the collection of cellular material from various surfaces. The new swab is derived from silica fibres with a chitosan coating. Six volunteers washed hands 15 minutes prior to touching each surface for a period of 15 seconds. The surfaces included plastic, glass, wood and metal. Foam, cotton, nylon-based and polyurethane swabs were compared to the chitosan-coated swab for the effective collection of deposited cellular material. Visualisation of latent cellular material was conducted using Di- amondTM nucleic acid dye allowing the monitoring of the amount of cellular material initially deposited on each substrate, then removed by the swabs, and how much remained on the substrate. Real-time PCR was then used to determine the amount of DNA obtained by each swab. Initial results indicate the newly developed fibre, nylon and polyurethane based swab were the most effective on plastic, metal and wood with only little differences in cellular and DNA collection recorded between all fibres on glass surfaces.

442 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P362 - COMPARISON OF TWO DNA EXTRACTION METHODS: PREPFILER® BTA AND MODIFIED PCI-SILICA BASED FOR DNA ANALYSIS FROM BONE Laila Hasap1, Wilaiwan Chotigeat1, Jintana Pradujkanchana2, Watee Asawutmangkul3, Thitika Kitpipit4, Phuvadol Thanakiatkrai4

1 Prince of Songkla University, Department of Molecular Biotechnology and Bioinformatics, Hat Yai, Thailand 2 Prince of Songkla University, Department of Pathology, Hat Yai, Thailand 3 Institute of Forensic Medicine, Subdivision of Biochemistry, Bangkok, Thailand 4 Prince of Songkla University, Department of Applied Science, Hat Yai, Thailand

Bones are important evidence often found in crime scene associated with mass grave disaster and missing person. Substantial fragmentation and intermixing of bones limit the use of morphological observation. Human individualization using DNA analysis can complement but the challenges are the PCR inhibitors contained and low amount of DNA in degraded bone. The selection of effective DNA extraction method that minimizes PCR inhibitors and maximizes DNA recovery thus is crucial. This study aimed to compare the efficiency of the currently modified PCI-silica and the latest forensic commercial kit developed for challenging bone sample: PrepFiler® BTA. The results showed that PrepFiler® BTA provided significantly higher median of DNA concentration (p < 0.05) and 95.3 % probability of higher number of allele compared to modified PCI-silica based method. The efficiency of PCR inhibitors removal showed that both extraction methods were comparable at minimizing PCR inhibitors compared to negative control. Our study could be one of the guideline for selection of the suitable method for DNA analysis from bone.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 443 P363 - COMPARISONS OF ALLELE SIZING BETWEEN GENOTYPING SOFTWARE Jillian Conte1

1 Keystone College, Biological and Physical Sciences, La Plume, USA

There is different genotyping software available for analyzing electrophoretic data. Some are purchased, while others are available as open source. Two commercially available software, Gen- eMarker HID (SoftGenetics) and GeneMapper (Thermo Fisher Scientific), use the Local Southern Method for sizing of alleles. This is the most commonly used algorithm in forensic DNA analysis. OSIRIS, a freely available software package that analyzed multiplex STR profiles departs from Southern methods, and instead uses the samples’ internal lane standard (ILS) and run-specific allelic ladder. This sizing method compares the timing of the ILS and ladder peaks to the sample peaks to size the alleles. Using an available database of electrophoretic samples1, the above genotyping software was used to analyze the samples with focus on the sizing of alleles. First genotypes were checked for concordance between the software. Data from single-source profiles constructed with two different STR chemistries and two different injection times were compiled and compared statistically among the three platforms. This work serves to expand our knowledge on the sizing of alleles in forensic samples and help us to better understand the data we are interpreting.

444 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P364 - COMPLETELY AUTOMATED INTERPRETATION OF REFERENCE SAMPLES Volker Weirich1

1 State Office of Criminal Investigation Mecklenburg-Vorpommern, Division 5- Department 54, Rampe, Germany

Inspired by fully continuous thinking we implemented a completely automated interpretation of reference samples in our lab. It’s based on a quite simple model, which includes DNA amount and degradation as well as backward and forward stutters. Current implementation is able to handle CE results of any number of replicates, even from different autosomal STR kits. Results, lessons learned from the first year’s usage and potential future extensions will be discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 445 P365 - CONSIDERING THE DNA TRANSFER ISSUES UNDER A RETROSPECTIVE ANALYSIS OF FORENSIC EXAMINATION Irina Perepechina1

1 Lomonosov Moscow State University, Department of Criminalistics of the Faculty of Law, Moscow, Russian Federation

Current knowledge concerning the DNA transfer makes it obvious that the data of forensic DNA identification should be assessed by both the investigator and the court in the situational context, taking into account the mechanism of trace formation. It especially concerns the examination of “touch” DNA. The perspectives of addressing activity-level questions are associated with using molecular approaches to forensic cell type identification (Sijen, 2015), as well as estimating the probabilities of possible scenarios based on the Bayesian methodology (Taylor et al., 2018). The most complex situation is the retrospective analysis of the forensic examination when further laboratory research is impossible due to the lack of the material, for one reason or another. For instance, the Art. 81 of the Code of Criminal Procedure of the Russian Federation suggests the possibility of the eliminating some types of physical evidence after the sentencing, that makes impossible their further examination. The situation may be aggravated by revealing some defects of the examination in the expert’s report, which in turn complicates the assessment. Such cases may happen in expert practice, hence the algorithm of resolving these issues should be outlined. The article considers the case with circumstances which could be important for interpreting results of DNA identification and, therefore, must be taken into account. The author discusses the model for evaluating data under retrospective analysis with a strong uncertainty factor.

446 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P366 - CRITICAL EVALUATION OF TOUCH DNA RECOVERY METHODS FOR FORENSIC PURPOSES Sophie Hartless1, Laura Walton-Williams1, Graham Williams1

1 Staffordshire University, Law, Policing and Forensics, Stoke-On-Trent, United Kingdom

Over the past decade there has been a significant increase in the number of submissions of ‘touch DNA’ evidence to forensic laboratories. Previous research has indicated that analysis of these samples produces poor results, with only 5–6 % of handled items generating a full profile (Quinones and Daniel, 2012). Published research, as well as case work review by forensic practitioners, has also indicated more consideration of how to improve the evidential value of ‘touch DNA’ samples is needed. Therefore, this research aims to critically evaluate low level DNA recovery methods in order to maximise efficiency for forensic identification purposes. Typical evidential items, such as plastic handled screwdrivers, aluminium cans, drinking glasses and wooden handles, were handled in a mock-operational trial. The deposited DNA was recovered from these items using a range of swabbing materials including cotton, polyester, nylon flocked, foam and viscose. These samples were then quantified using human specific quantitative PCR and profiled using AmpFLSTR™ NGM SElect™. The DNA quantity and quality were compared and a statistically significant difference was found to be present between recovery methods. These findings will be presented and discussed in this paper. Further to this, the findings of this research will be evaluated to determine the optimal recovery method for differing surface types. The way in which the DNA interacts with the surfaces and the swabbing materials will also be evaluated to determine the impact this has upon the quality of the profiles produced. This research will inform best practice for the recovery of low level DNA samples from forensic exhibits and can influence the ISO validation procedures for crime scene examination processes (ISO17020).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 447 P367 - DEALING WITH LOW AMOUNTS OF DEGRADED DNA: EVALUATION OF SNP TYPING OF CHALLENGING FORENSIC SAMPLES BY USING MASSIVE PARALLEL SEQUENCING Chiara Turchi1, Valerio Onofri1, Filomena Melchionda1, Carla Bini2, Carlo Previderè3, Eugenia Carnevali4, Carlo Robino5, Solange Sorçaburu-Ciglieri6, Giorgio Marrubini7, Susi Pelotti2, Paolo Fattorini6, Adriano Tagliabracci1

1 Polytechnic University of Marche, Department of Biomedical Sciences and Public Health, Ancona, Italy 2 University of Bologna, Department of Medical and Surgical Sciences, Bologna, Italy 3 University of Pavia, Department of Public Health- Experimental and Forensic Medicine, Pavia, Italy 4 University of Perugia, Section of Legal Medicine and Forensic Science - S. Maria Hospital, Terni, Italy 5 University of Turin, Department of Public Health Sciences and Pediatrics, Turin, Italy 6 University of Trieste, Department of Medicine, Surgery and Health, Trieste, Italy 7 University of Pavia, Department of Drug Sciences, Pavia, Italy

MPS technology provides advantages for the forensic analysis of degraded DNA samples, due to its capability to genotype a large number of SNPs in a single assay. The Precision ID Identity Panel is designed to detect 90 autosomal SNPs together with 34 Y-SNPs. To improve our understanding over the performance of this panel in casework analysis, a collaborative exercise was set up. The primary aims of the present study were i) to investigate the effectiveness of the panel with low amounts of degraded samples; ii) to optimize the analytical conditions of such samples. Thus, six laboratories collected a large set of forensic samples (n=87) including bone remains, cadaveric blood, cadaveric muscle, fingernails, FFPE tissues, touch DNA together with a set of artificially degraded DNAs. The availability of a reference sample (either “same donor” sample or “first degree relative” sample) was an essential recruitment criterion. After assessing the degradation index by Quantifiler™ Trio DNA Quantification Kit, the libraries were prepared by using 0.012–1.0 ng of template. Different number of PCR cycles (from 21 to 26) was tested. Dilutions down to 12 pg of the 2800M DNA were used as controls. Overall, a total of 256 MPS assays were performed by Ion Torrent PGM platform. The results shown that, in spite of decreased coverages, full or almost full profiles can be obtained even in highly degraded samples when the amount of template range from 0.1 to 1.0 ng. Finally, the increment of the number of PCR cycles does not seem to provide an improvement in typing results of low amounts of degraded samples as, in front of higher number of typed loci, higher frequencies of artefacts (mainly allele drop-out events) leading to mistyping are found at 25–26 cycles.

448 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P368 - DEGRADED DNA TYPING HIGHLY IMPROVED BY A CODIS CORE + ESS MEGAPLEX BASED ON SUPERPRIMERS Martin E. Mautner1, Mariela Caputo2, Tatiana Bengochea1, Daniel Corach2

1 Biodynamics SRL, R&D Laboratory, Buenos Aires, Argentina 2 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Genética Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG. CONICET, Buenos Aires, Argentina

Multiplex amplification of short tandem repeats (STR) markers followed by capillary electrophoresis (CE) is a widely used technique for genotyping DNA samples. Unfortunately, this method often provides partial or null profiles for the degraded DNA normally found in forensic, aged and archeological samples. This downside arises from the need to amplify several long amplicons in order to allocate multiple markers into the CE spectrum. Since degraded DNA is fragmented, primers designed to anneal far apart usually fail to amplify the longer sequences. We hereby show that single-stranded DNA (ssDNA) polynucleotides can be used as primer sur- rogates in multiplex PCR assays. In that way these long primers can de designed to anneal next to the repeat sequences while at the same time they themselves contribute to create longer amplicons. They act in a similar fashion to the mini-STRs primers, with the additional advantage of providing PCR products with suitable sizes for covering the full CE range. We have combined several oligonucleotide and polynucleotide primers (up to 200 nucleotides, referred to as superprimers) in a multiplex PCR cocktail to allow the allele identification of all the Combined DNA Index System (CODIS) core and European Standard Set (ESS) loci in a single reaction. Our results suggest that multiplex assays containing superprimers provide higher genotyping information from degraded DNA samples than the standard multiplex STR kits.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 449 P369 - DETECTION OF CELLULAR MATERIAL WITHIN HANDPRINTS Piyamas Kanokwongnuwut1, Paul Kirkbride1, Adrian Linacre1

1 Flinders University, College of Science and Engineering, Adelaide, Australia

A novel technique for the visualisation of cellular material has been published harnessing a fluorescence nucleic acid dye “Diamond dye” (DD) in combination with a digital microscope “Dino-lite”. This technique can effectively detect cellular material transferred by touch allowing targeting collection of human DNA. Studies on touch DNA have focussed on transfer from fingertips. Here we report on the visualisation of cellular material transferred via twenty different positions on entire handprints. Three volunteers (one high, one intermediate and one poor shedder) were asked to press their entire hands on a plastic surface with medium pressure. DD was applied to the area and the presence of cellular material recorded using a Dino-lite microscope. The location and amount of cellular material was recorded using software developed in-house. Scoring of transferred cellular material was observed from 3 locations of each finger, 3 from the thumb, and 5 from the palm to give 20 positions in total per hand-print. Cellular material within 5 separate frames at each of the 20 positions was recorded and as all tests were performed in triplicate, in total there was 1,800 observed frames. This extensive study allows the accurate transfer of cells from different parts of the whole hand to be monitored and shows which areas shed the greatest, or least, amount of cellular material. This simple process can act as a guide for DNA collection from items held within the hand rather than only fingertip, such as: weapons, knives, and steering wheels.

450 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P638 - DEVELOPING A DNA METHYLATION-BASED MULTIPLEX SNP ASSAY FOR THE IDENTIFICATION OF SEMEN IN MIXED STAINS Bowen Xie1, Feng Song1, Yanyun Wang2, Shuangshuang Wang1, Yun Huang1, Haibo Luo1, Yingbi Li1

1 West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Forensic Genetics, Chengdu, China 2 West China Second University Hospital- Sichuan University, Laboratory of Molecular Translational Medicine, Center for Translational Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children Sichuan University, Ministry of Education, Chengdu, China

In forensic investigations, especially in sexual assault cases, biological samples are often mixed stains of different types of body fluids. Highly uneven proportions DNA of male and female makes the detection for stains more difficult. Recently, DNA methylation was proposed to be a promising maker for body fluid identification, and successfully applied to mixed DNA samples. In this study, 10 semen-specific sites were selected from the dataset GSE59509. Methyl- ation-specific polymerase chain reaction and SNaPshot technology were then used to detect the genotype of SNPs in these sites. The results showed that 10-plex SNP typing system was specific in semen samples. In mixed stains, when semen and vaginal fluid in volume ratios of 1:25, the analyses for 10-plex SNP showed that the semen derived genotype could be determined correctly. Meanwhile, unambiguous profiles can be obtained when the input DNA was 0.25 ng. In addition, the 10-plex assay may be useful in the identification of semen even in the degraded samples and complex mixtures.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 451 P370 - DEVELOPMENT AND VALIDATION OF 21-PLEX STR PANEL Natalya Khodeneva1, Natalya Ermakova1, Elmira Larionova1

1 NEARMEDIC PLUS Ltd, Laboratory of molecular biology developments, Moscow, Russian Federation

Short tandem repeat (STR) analysis is the most common and informative type of DNA profiling widely used in paternity testing, missing persons cases and forensic applications. In this study we report the development of the in-house STR panel that contains 21 loci: the 13 original CO- DIS loci with 4 non-overlapping loci from the expanded ESS, as well as SE33 and D6S1043, one Y-STR DYS391 and the sex determining marker Amelogenin. Primers for each locus were designed in-house, labelled with five different fluorescent dyes, tested in single PCR reactions and combined into a multiplex mix. Allelic ladder of 256 fragments was developed, and each fragment was sequenced to confirm the repeat structure. After optimization of the master mix and PCR conditions, we were able to amplify DNA directly from Whatman FTA cards as well as from buccal swabs treated with SwabSolution™ Kit, Prep-n-Go™ Buffer or Chelex® 100. Developmental validation studies of the novel 21-plex assay were performed using ProFlex™ PCR System, Veriti™ Thermal Cycler and GeneAmp™ PCR System 9700 for DNA amplification. Capillary electrophoresis was performed on Applied Biosystems 3500 or 3500xL Genetic An- alyzers. The sensitivity study showed accurate and balanced genotyping of extracted DNA in a range from 0.125ng to 2ng per reaction. The genotype concordance study showed 100 % concordance of the 21-plex assay in comparison with VeriFiler™ Express PCR Amplification Kit. Additionally, we were able to correctly detect and report all unknown alleles within the GEDNAP Proficiency Tests 56 & 57 using the developed 21-plex STR panel. To summarise, the novel STR panel is a valuable alternative to the current commercial kits for human DNA identification including forensic analysis, paternity testing and research use.

452 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P371 - DEVELOPMENT AND VALIDATION STUDY OF THE monSTR FORENSIC IDENTITY PANEL, A MULTIPLEX STR KIT FOR MASSIVELY PARALLEL SEQUENCING Janine Silvery1, Sebastian Ganschow1, Helena Siemens1, Peter Wiegand2, Carsten Tiemann1,3

1 LABCON-OWL Analytik- Forschung und Consulting GmbH, Forensic Genetics, Bad Salzuflen, Germany 2 University Hospital of Ulm, Institute of Legal Medicine, Ulm, Germany 3 University of Applied Science, Faculty of Engineering and Mathematics, Bielefeld, Germany

The application of massively parallel sequencing (MPS) in forensic genetics enables high-resolution genotyping for characterization of biological evidence. Furthermore, the MPS technology far exceeds the multiplexing capacity of current forensic markers which may be typed in the same assay. We described the systematic development of the 21‑plex STR panel monSTR, which was designed for high-fidelity forensic genotyping on the Illumina MiSeq platform. The selection of STR markers adapts on the expanded European Standard Set (ESS), including the highly polymorphic locus SE33, for compatibility with existing forensic DNA databases. Moreover, the panel performance was validated according to the SWGDAM guidelines. Studies herein comprised a series of experiments that evaluated concordance, repeatability, sensitivity of detection, mixture analysis, species-specificity, and the ability to analyze case-type samples. A set of statistical metrics was developed to measure the assay performance, including read on-target ratio, genotype accuracy, inter-locus balance, heterozygosity balance, and signal-to-noise ratio. Results showed that inter‑run and intra‑run repeatability indicated high accuracy and precision. Full profiles could be obtained using 62.5 pg of input DNA with proper inter-locus balance and on-target ratio; more than 75 % of alleles were correctly called with 7.8 pg input DNA. Case type samples were fully concordant with CE-based methods and satisfying allele coverage ratio for SE33. Results also suggested that the minor contribution could be precisely calculated based on minor component allele read counts. It could be demonstrated that the monSTR panel is a valid tool for forensic routine DNA typing using the MPS technology.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 453 P372 - DEVELOPMENT OF A 40 LINKED AUTOSOMAL STRs PANEL USING MASSIVELY PARALLEL SEQUENCING Qingwei Fan1, Yan Wang2, Jing Zhao3, Feng Cheng1, Mengchun Wang1, Chen Li4, Chuguang Chen4, Yacheng Liu5, Gengqian Zhang1, Jiangwei Yan1

1 Shanxi Medical University, School of Forensic Medicine, Taiyuan, China 2 The Seventh Medical Center of PLA General Hospital, BaYi Children’s Hospital, Beijing, China 3 Chinese Academy of Sciences, Beijing Institute of Genomics, Beijing, China 4 Beijing Microread Genetics Co.- Ltd, Beijing Microread Genetics Co.- Ltd, Beijing, China 5 Beijing Tongda Shoucheng Institute of Forensic Science, Beijing, China

Short tandem repeats (STRs) due to their polymorphic nature are well-studied and routinely used in forensic DNA typing. If we take linkage and recombination fraction into consideration, linked autosomal STRs (LaSTRs) can provide more meaningful information than independent makers in some complex kinship analysis. However, most commercial STR kits only have been included independent STR loci except for a few LaSTR. In this study, 11 groups of 40 LaSTRs loci were selected from Rutgers Map, which were located on different chromosome, including two groups of five loci, three groups of four loci and the remaining six groups of three loci, respectively. Multiplex amplification technologies have used to develop a panel including the selected 40 LaSTRs, which amplicons range from 153bp to 256bp sequencing by Illumina MiSeq FGx sequencing platform. A population study with 102 unrelated individual from northern Han of Chi- na was performed using this panel. The corresponding allelic frequencies ranged from 0.005 to 0.589. Expected heterozygosity, polymorphic information content, power of discrimination and power of exclusion of the 40 STR loci were from 0.656 to 0.878, from 0.592 to 0.861, from 0.653 to 0.878 and from 0.393 to 0.745, respectively. These results stated that the LaSTRs panel developed by our study may be a meaningful tool for complex kinship analysis.

454 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P373 - DEVELOPMENT OF A MODIFIED PROTOCOL FOR EZ1 (QIAGEN) AUTOMATED DNA EXTRACTION FROM HUMAN REMAINS Anna Barbaro1, Patrizia Cormaci1, Angelo La Marca1

1 Studio Indagini Mediche E Forensi SIMEF, Forensic Genetics, Reggio Calabria, Italy

EZ1 Advanced XL is designed to purify automatically nucleic acids from a wide variety of forensic samples: different DNA extraction protocols are pre-loaded into special EZ1 Advanced XL DNA Investigator Cards. The present paper focuses on the modification of the protocol already available for DNA extraction from human remains. To develop the procedure we selected several bones and teeth collected since 1 to 50 years after death and previously subjected to other extraction methods. Our findings showed EZ1 modified DNA extraction protocol for bones/teeth allows to recover DNA, free of inhibitors and in enough quality/quantity for downstream application. This efficacious method in combination with sensitivity PCR multiplexes produced from all tested samples high quality reliable STRs profiles.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 455 P374 - DEVELOPMENT OF INVESTIGATOR 26PLEX QS, A NEW MULTIPLEX PCR KIT FOR GLOBAL STR ANALYSIS Miroslav Vranes1, Margaretha König1, Stefan Cornelius1, Annika Kohns1, Mario Scherer1, Ralf Peist1, Anke Prochnow2, Keith Elliott3

1 QIAGEN GmbH, Research and Development, Hilden, Germany 2 QIAGEN GmbH, Product Management, Hilden, Germany 3 QIAGEN GmbH, Marketing, Hilden, Germany

We developed an assay co-amplifying 25 markers including the expanded CODIS loci. The assay uses a 6-dye technology in order to keep the amplicon length of markers short while at the same time avoids overlapping of markers. The kit is designed for purified DNA from casework and reference samples, and can also be used for direct amplification of reference samples like blood or buccal cells on FTA or swabs. Furthermore, the kit contains the Quality Sensor System that is useful for evaluating the amplification efficiency. It indicates if the reaction has worked in general and furthermore allows discriminating between the presence of inhibitors or DNA degradation as a cause for the typical ski slope effect observed in STR profiles of such challenging samples. This information can be used to choose the most appropriate rework strategy. The assay is based on a new PCR chemistry FRM3.0 that ensures robust and fast PCR amplification with improved inhibitor resistance and easy handling.

456 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P375 - DEVELOPMENTAL VALIDATION OF A PROBE CAPTURE NGS SYSTEM FOR ANALYSIS OF WHOLE MITOCHONDRIAL GENOME OF FORENSICALLY CHALLENGING SAMPLES Shelly Shih1, Rachel Gordon2, Daniela Cuenca2, George Sensabaugh3, Henry Erlich4, Cassandra Calloway4

1 Children’s Hospital Oakland Research Insitute, Calloway Lab, Oakland, USA 2 California Department of Justice, Jan Bashinski DNA Laboratory, Richmond, USA 3 University of California- Berkeley, School of Public Health, Berkeley, USA 4 Children’s Hospital Oakland Research Institute, Calloway Lab, Oakland, USA

Mitochondrial (mt/mito) DNA analysis has become a particularly useful tool for testing samples containing insufficient nuclear DNA for conventional STR analysis. In highly compromised samples, DNA degradation can reduce the presence of two intact primer binding sites on the template DNA fragments, often resulting in failure of PCR amplification of the targeted genetic markers. To allow the analysis of these challenging case-type samples, we have developed and validated a probe capture next-generation sequencing (NGS) system for target enrichment of mtDNA from highly degraded samples. Our method utilizes biotinylated DNA probes in a tiling pattern across the mito-genome to hybridize to all mitochondrial haplotypes and NGS to achieve ample sequence read depth and digital read counts to provide sensitive and quantitative detection of minor sequences at mixed base positions. We have completed a developmental validation of the probe capture NGS system following the standard tests in the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines. We demonstrated that the probe capture NGS system is sensitive, robust, precise, and accurate for sequencing of the whole mito-genome from reference and simulated case samples. Our data showed that this system is sensitive in detecting minor contributor sequences in a 95:5 two-person mixture at input DNA of 1 ng. This system is capable of generating valuable sequence data for case-type samples including shed hairs, cut hairs, solid tissues, and touch DNA recovered from spent brass cartridges.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 457 P376 - DEVELOPMENTAL VALIDATION OF THE CASEWORK DIRECT KIT FOR EFFICIENT SCREENING OF SEXUAL ASSAULT AND TOUCH DNA SAMPLES Erica Graham1, Mary Loten1, Jonelle Thompson1, Jon Drobac1, Anupama Gopalakrishnan1, Jeanne Bourdeau-Heller2, Robert McLaren2, Lotte Downey1, Douglas Storts2

1 Promega Corporation, Genetic Identity, Madison, USA 2 Promega Corporation, Research and Development, Madison, USA

Standard extraction methods for forensic samples are often time-consuming and include multiple wash steps which can introduce opportunities for DNA loss. The Casework Direct Kit, Cus- tom provides a simple, fast DNA extraction method without purification steps. Integration of the Casework Direct Solution Kit into a laboratory workflow scheme provides a rapid, cost effective means to generate high quality STR profiles from precious, low-abundance samples with minimal hands on time. The lysate generated from the Casework Direct Kit, Custom is compatible with Promega quantification and amplification systems. The experiments described in this poster address the federal standards and guidelines established by the FBI Quality Assurance Standards (QAS) for Forensic DNA Testing Laboratories and the Scientific Working Group on DNA Analysis Methods (SWGDAM). The experiments will examine sensitivity, reproducibility, stability, mixtures, contamination and non-probative sample criteria. These analyses demonstrate that the Casework Direct Kit, Custom, is a suitable, accurate and reproducible method for the rapid isolation of DNA.

458 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P377 - DIAMOND™ NUCLEIC ACID DYE AND MICRO FLOQ® DIRECT SWABS FOR FORENSIC CASEWORK Carsten Proff1, Julia Kolb1, Natalie Schury1, Ingo Bastisch1

1 Bundeskriminalamt - BKA, KT31 - DNA Analysis, Wiesbaden, Germany

DNA-analysis of touch evidence gains relevance in everyday casework. Nowadays, a faster delivery of significant results becomes more and more important. With a direct PCR approach, some workflow steps can be skipped. Furthermopre, staining opportunities for traces on objects of evidence exist and may improve search and detection of touch DNA evidence. Diamond™ Nucleic Acid Dye und MicroFloq® Direct Swabs were tested on their suitability in forensic casework in comparison to standard routine methods. Results on fluorescent staining of different items and surfaces as well as typing results on direct PCR of different sample types are presented. Limitations and advantages of the two methods are described.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 459 P378 - DIFFERENTIAL EXTRACTION METHOD AS A GOLDEN STANDARD IN ANALYZING OF SEMEN STAINS IN SEXUAL‑ASSAULT CASES Renata Jankova1, Zlatko Jakovski1, Robert Janevski2

1 University St.Cyril and Methodius, Medical Faculty, Institute of Forensic Medicine, Criminalistic and Medical Deontology, Skopje, Macedonia the former Yugoslav Republic 2 Ministry of Internal Affairs of the Republic of Macedonia, Forensic Department, Skopje, Macedonia the former Yugoslav Republic

In the investigation of sexual-assault cases different analysis of collected biological samples from the victim and the crime scene are applicable, depending of the results of the presumptive tests for sample characterization. DNA testing of semen stains can be performed in different ways. Differential extraction method is a golden standard for analysis of sexual assault mixture samples of sperm cells and victim epithelial cells, allowing straight forward interpretation of perpetrator’s DNA profile. We have to keep in mind that it is not applicable in about 1,9 % of semen stains found in the casework that shows condition of azoospermia. We present two cases where differential extraction method was decisive approach in analysis of sexual assault evidence in the trial process.

460 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P379 - DIRECT PCR OF BLOODSTAINS COLLECTED FROM DECEASED INDIVIDUALS FOR IDENTIFICATION PURPOSES Zoe Bowman1, Valerie Chahin Atallah1, Dadna Hartman1,2

1 Victorian Institute of Forensic Medicine, Molecular Biology, Southbank, Australia 2 Monash University, Department of Forensic Medicine, Melbourne, Australia

Direct PCR provides advantages over traditional DNA processing by removing the DNA extraction and quantification steps, thus reducing sample processing time. Such advantages are invaluable in disaster victim identification (DVI) and other cases where DNA identification of a deceased is required. Where possible we collected bloodstains on Copan NUCLEIC-CARD™ in cases where DNA identification of a deceased is required. An attempt was made to develop a direct PCR processing pipeline for bloodstains collected from deceased individuals. Initial testing with GlobalFilerTM Express using the manufacturer recommended 1.2 mm sample punches showed significant inhibition in many samples, with 20 (77 %) of the 26 samples tested resulting in full profiles. This first pass success rate is insufficient for our purposes. Another concern were the unusually high baseline artifacts seen in relative proportion to the peak heights obtained. The GlobalFilerTM casework kit was also trialled with 1.2 mm sample punches which demonstrated a cleaner baseline but increased inhibition. To reduce the amount of inhibition, the amount of sample added to the PCR reaction was halved which resulted in 4 of the previously unsuccessful samples producing a full profile using both GlobalFilerTM Express as well as less inhibition effects in other samples tested. GlobalFilerTM casework was also successful with a halved input however more inhibition was observed when compared to GlobalFilerTM Express. The 2 remaining unsuccessful samples that failed to produce any results had, however, previously given results with traditional profiling. Pre amplification dilution of samples had some success in producing profiles from these samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 461 P380 - DIRECT PCR USING MICROFLOQTM DIRECT SWABS WITH A MODIFIED QIAGEN INVESTIGATOR 24PLEX GO! PROTOCOL FROM DECOMPOSING HUMAN REMAINS FOR DVI APPLICATIONS Coral Loockerman1, Sheree Hughes-Stamm1,2, Rachel Houston1

1 Sam Houston State University, Department of Forensic Science, Huntsville, USA 2 University of Queensland, School of Biomedical Sciences, Brisbane, Australia

Disaster victim identification (DVI) often relies on rapid identification of decomposing human remains, possibly in remote areas without access to refrigerated storage facilities. Collection of biological material using swabs may prove easier, more efficient, and more amenable to storage in harsh conditions. microFLOQ™ direct swabs have been identified as a potential alternative for more rapid collection and processing of DNA in forensic and DVI situations. 4N6FLOQSwabs™: Genetics and microFLOQ™ direct swab were used to collect DNA from red muscle via an incision in the arm or leg of a decomposing human cadaver. Traditional DNA processing with the Genetics swabs was compared to a direct amplification strategy using the microFLOQ™ swab coupled with the Investigator 24plex QS GO! Kit. The direct amplification strategy was optimized by pre-treating the swab (washing, vortexing, lysis) prior to amplification and slightly modifying the cycling parameters. As an alternate method, the microFLOQ™ swabs were used to sub-sample DNA stored on the Genetics swabs. STR success rates of traditional and direct PCR method were comparable but were highly dependent on the stage of decomposition and the sample location. The Quality Sensor (QS) markers were used to assess sample quality. Interestingly, the QS markers indicated that even after pre-washing the microFLOQ™ swabs, there was still inhibition present in the amplification for swabs that were dried overnight. However, when the microFLOQ™ swabs were processed within hours of swabbing or used to sub-sample, there was no inhibition indicated by the QS markers and profile completeness improved. Overall, microFLOQ™ swabs in conjunction with the GO! Kit facilitated direct processing in the laboratory from decomposing remains.

462 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P381 - DNA EXTRACTION FROM DIFFERENT SKELETAL HUMAN REMAINS WITH DEMINERALIZATION PROTOCOLS, ORGANIC EXTRACTION AND USING SILICA Jose Andres Carrasco Raimrez1, Pantoja Astudillo Jaime2, Ojeda Pugin Viviana2, Honorato T Josefina2, Figueroa Cecilia2, Solar Henry2, Morales G Carmen2

1 Servicio Medico Legal, Bioquimica y Criminalistica, Santiago, Chile 2 Servicio Medico Legal, Unidad de Genetica Forense, Santiago, Chile

Many types of bones and skeletal human remains are a good source of DNA, useful in many cases for forensic analysis, especially when the soft tissue is severely decomposed or damaged. There are different methods for the extraction of DNA from bone remains, including samples greater than 20 years old. The 50 Bone samples were pulverized to a fine powder (0.8 g) and obtain 0.5 – 0.02 ng/ml of DNA concentration . Exists many protocols to works bones but they used separately. In our study we compare the efficiency the demineralization, organic extraction an silica used combined. The results prove that combined protocols its very useful to obtain to recovery DNA concentration to suitable to obtain full autosomal profile.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 463 P382 - DNA POLYMERASE PERFORMANCE ON THE DEGRADED HUMAN DNA SAMPLES Kyungyong Kim1

1 College of Medicine- Chungang University, Ancient Human DNA Institute, Seoul, Republic of Korea

Human DNA from tombs, soil, or ancient burials are highly degraded. Especially very old and severely fragmented human DNA samples are common in the forensic studies. Despite many various methods and efforts to identify the DNA status, it is often found out to be unsuccessful due to the poor quality and quantity of the DNA samples. We have extensively evaluated the most used DNA polymerase for the successful pcr of severely fragmented human DNA samples. We have tried to study various DNA polymerases. We found DNA polymerases in the market, which included enzymes that are reportedly effective for pcr-inhibitory samples. We could give comments that the several DNA polymerases are more efficient for higher probability of success pcr that could give the desired results from highly degraded human DNA samples.

464 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P383 - DNA PROFILES OBTAINED FROM URINE IN SNOW Charlotte Dufva1, Emilia Karlsson1, Christina Forsberg1, Ricky Ansell1,2

1 National Forensic Centre, Swedish Police Authority, Linköping, Sweden 2 Department of Physics- Chemistry and Biology IFM, Linköping University, Linköping, Sweden

Urine can be a potential important source of evidence when occurring at crime scenes. In case of outdoor scenes including snow a yellow colour could indicate the existence of human urine. However, in urine, cell destruction and DNA degrading can occur, which makes it a challenging material. In an effort to investigate urine as a crime scene sample we have evaluated protocols for analysing DNA from urine in snow. Two different tests were performed with a small and a larger volume of urine dispensed on snow in 50 mL tubes. The tubes were put into a freezer to mimic winter conditions. A Urine Preserva- tive (Norgen Biotek Corporation) was added to some of the samples. DNA profiles (PowerPlex® ESX 16 Fast System (Promega)) were compared between samples extracted using a Urine DNA Isolation Kit (Slurry Format) (Norgen Biotek Corporation) and samples extracted with a Chel- ex-based method. In addition, a test was performed with the aim to mimic a potential crime scene. Several men urinated in snow, that was collected a couple of hours later. These samples were handled as described above. With a small volume of urine the best quality DNA profiles were obtained using the Urine DNA Isolation Kit without the Urine Preservative. When a larger volume of urine was handled and in the crime scene setup, there were no clear difference between the two extraction methods. Instead, the variation observed was between individuals. In conclusion, it is possible to obtain DNA profiles from urine in snow.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 465 P384 - DNA RECOVERY FROM SERATEC IMMUNOCHROMATOGRAPHIC TESTS Jillian Conte1, Jordyn Olsen1, Loretta Owusu1, Gabriela Roca2

1 Keystone College, Biological and Physical Sciences, La Plume, USA 2 SERATEC GmbH, SERATEC GmbH, Goettingen, Germany

Immunochromatographic tests strips are being used increasingly to presumptively identify bodily fluids of forensic interest such as blood, semen, and saliva. The forensic sample would travel across a membrane and interact with embedded antibodies to indicate the presence of a bodily fluid antigen of interest, and then the testing cassette gets discarded. Commonly, forensic samples are of limited quantities. In the practice of conserving limited samples, it would be ideal to be able to recover the genetic material deposited on these testing membranes. The goal of this research was to determine if genetic material can be recovered from immunochromatographic test strips following use. The SERATEC® PSA and Amylase Tests had semen and saliva samples, respectively, deposited and analyzed. The testing membrane was then removed from the cassette and DNA extraction methods performed. Equivalent volumes of sample that were not analyzed by the immunochromatographic test strips underwent DNA extraction. The samples were quantified and statistical calculations performed to determine retention of DNA by the membrane. The method of eluting DNA from testing strips should be optimized for future work.

466 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P385 - DNA TESTING OF TOUCH EVIDENCE ON A HAND TOWEL Katsuya Honda1, Keishi Umino1, Hisanori Muramatsu1, Shigeru Akanuma1, Yayoi Iwabuchi1, Fujio Ishizawa1, Yukiko Sugano1

1 University of Tsukuba, Department of Legal Medicine, Tsukuba city, Japan

Recent progress in DNA technology has enabled DNA detection from poor-quality samples or trace amounts of cells. Here, we examined the possibility of detecting trace amounts of DNA by simulating a scenario in which an assailant attempted to push a hand towel into the victim’s mouth and suffocate them. Skin secretion from the assailant and saliva from the victim were assumed to be attached to the towel. Materials and Methods: We obtained part of the towel that the assailant had touched and extracted DNA from it. We included 10 assailants for one victim and performed tests for each of them. Organic DNA extraction was carried out using phenol/chloroform with lysis buffer in a 50-ml centrifuge tube. Extracted DNA was used as a template for DNA analysis. DNA testing was conducted using three methods: 1) STR typing using AmpFlSTR Identifiler PlusKit (Thermo Fisher); 2) Mitochondrial DNA (D-loop) sequencing using Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher); and 3) Hyper-sensitive STR and SNP analysis using MiSeq FGx System(Verogen). Results and discussion: After comparison with a reference sample, a mixed profile of the assailant and the victim, and a single profile of the assailant were obtained using each of these three methods. Among these methods, the Identifiler detection had a high rate of detection for the assailant–victim mixed profile. Moreover, the MtDNA detection had a high rate of detection for the assailant only. The FGx method had the highest detection power for mixed alleles. All testing proved effective for examining touched samples in actual forensic cases. Detection of DNA was possible from a towel sample touched by an assailant, using each of these methods. Therefore, we should use these methods appropriately depending on the nature of the criminal case.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 467 P386 - DNA TYPING FROM SKELETAL REMAINS USING GLOBALFILER® PCR AMPLIFICATION AND INVESTIGATOR® 24PLEX QS KITS Dragana Zgonjanin1, Dalibor Nedić2, Rashed Alghafri3, Stojan Petković1, Radenko Vuković1

1 Clinical Center of Vojvodina, Institute of Forensic Medicine, Novi Sad, Serbia 2 Clinical Center of Banja Luka, Institute of Forensic Medicine of Republic of Srpska, Banja Luka, Bosnia and Herzegovina 3 Dubai Police G.H.Q., General Department of Forensic Sciences and Criminology, Dubai, United Arab Emirates

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Inves- tigator® 24plex QS kit and GlobalFiler® PCR Amplification kit, previously analyzed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown result in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain STR DNA profiles in 98 % of the bones.

468 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P387 - EFFECT OF PHI29 POLYMERASE-BASED MULTIPLE STRAND DISPLACEMENT WHOLE GENOME AMPLIFICATION ON THE PROPORTION IN DNA MIXTURE Qiang Zhu1, Ting Fang1, Yijun Zhou1, Yiwen Yang1, Yueyan Cao1, Qiuyue Wang1, Yuguo Huang1, Yuhan Hu1, Xiaogang Chen2, Yufang Wang3, Ji Zhang1

1 West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Department of Forensic Genetics, Chengdu, China 2 West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Department of Forensic pathology, Chengdu, China 3 West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Department of Pathophysiology, Chengdu, China

Multiple displacement amplification (MDA), an isothermal whole genome amplification method, has been applied extensively for its long amplification products and low bias. While the MDA method hasn’t further used in forensic science. MDA was known as resulting preferential amplification and allelic drop-out as dealing with low template amounts (<100 pg). But in the forensic mixture issues, MDA was reported to improve enlarging the proportion of minor DNA in mixture. To validation the application of MDA in DNA mixture, we used new commercial MDA kit, made different mixture ratios from 1:5 to 1:40, then compared mixture proportion and locus drop-out rate of minor DNA between PCR-STR with MDA and PCR-STR without MDA. Meanwhile, we validated the reproducibility of PCR-STR with MDA in different ratios. The results showed sufficient improvements in locus drop-out rate and mixture proportion of minor DNA after PCR-STR with MDA. Reproducible and complete minor DNA STR allele in different mixture ratios showed a fine reproducibility of MDA method. MDA would be an auxiliary method for dealing with forensic mixture issue.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 469 P388 - EFFICIENCY OF CASEWORK DIRECT KIT FOR EXTRACTION OF TOUCH DNA SAMPLES OBTAINED FROM CARS STEERING WHEELS Cintia Fridman1, Fernanda de Toledo Gonçalves1, Daniela de Oliveira Francisco1

1 Universidade de São Paulo, Departamento de Medicina Legal- Ética Médica e Medicina Social e do Trabalho, Sao Paulo, Brazil

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplifica- tion was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7 % and 59.9 %, respectively) than those extracted with DNA IQ™ (60.4 % and 38.1 %, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™. Financial Support: Promega Corporation, FAPESP(16/14355-3,17/06484-0),HCFMUSP/LIM40.

470 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P389 - EFFICIENT PRESERVATION OF DNA EXTRACTED FROM BLOOD IN FTA CARDS BY CHELEX METHOD German Burgos Figueroa1, Rodrigo Flores-Espinoza2, Viviana Alejandra Ruiz Pozo3, Irina Villacrés Granda4

Chelex resin has been developed and widely used for extracting DNA from different forensic-type samples. It is known that DNA extracted by this method is less prone to contain PCR inhibitors and it can be only stored for 3 to 4 months. No studies have shown the conservation of the extracted samples over time in samples from buccal swabs and blood in FTA card. This situation is a limitation in the population genetic studies for conservation of long-time lasting reference samples. For this study, we tested three DNA extraction methods: Chelex 10%-Protein- ase K (CHK), Phenol Chloroform (PC) and Chelex 10%-Proteinase K purified with Phenol Chlo- roform (CHK+PC), in two different kind of samples: blood stored in FTA cards and buccal swab samples. Beta-Actin gene (292 bp) was amplified by PCR at 0, 4, 8 months; STR profiles and amplification of mitochondrial DNA (1200 bp) were done at 8 months to check the DNA quality. Blood samples in FTA card extracted with CHK+PC method and buccal swab samples extracted with PC method showed amplification at 0, 4 and 8 months. In the other hand, both samples extracted with CHK method showed amplification only at 0 and 4 months and no profiles at 8. Samples that were kept for 8 months and previously extracted with PC and CHK+PC methods were successfully amplified showing a 1200 bp size and readable STR profiles. The conservation of the sample after DNA extraction depends mostly on the type of sample and the method used for PC method and CHK+PC method showed to be the better for sample conservation. These results suggest the importance of choosing the DNA extraction method that guarantees the conservation of reference samples or valuable forensic DNA specimens for further studies or forensic tests.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 471 P390 - ESTABLISHING STR AND IDENTITY SNP ANALYSIS THRESHOLDS THAT ENABLE RELIABLE INTERPRETATION AND PRACTICAL IMPLEMENTATION FOR MPS GENOMIC DNA CASEWORK Meghan Didier1, Susan Welti2, Jessica Skillman2, Stephanie Hickey2, Cydne Holt1, Melissa Kotkin1, Michelle Peck3, Kathryn Stephens1

1 Verogen Inc., Forensic Science and R&D, San Diego, USA 2 District of Columbia Department of Forensic Sciences, Forensic Biology Unit, Washington DC, USA 3 International Commission on Missing Persons, Validation and Development, The Hague, Netherlands

Massively parallel sequencing (MPS) evaluation by the global forensic community has demonstrated immediate advantages and forthcoming, versatile capabilities to aid missing persons and criminal casework. As a result, an increased number of operational casework laboratories are executing validation geared toward informing MPS interpretation guidelines and SOPs for routine implementation. As with CE-based data, establishing analysis thresholds that enable reliable interpretation within the limitations of a system is a pivotal decision point. We present an empirical data-driven approach for determining analytical and stochastic thresholds (AT, ST) for the 152 STR (auto, Y and X) and identity SNP (iSNP) loci targeted in the ForenSeq DNA Signature assay. While the methods utilized mimic CE threshold determination, fundamental distinctions specific to MPS data are addressed. Key considerations include relative and fixed read count values as well as interlocus balance expectations when increasing numbers of markers and/or marker types are multiplexed together in a single amplification. Internal data as well as data from collaborating laboratories will be presented. For AT assessment, sensitivity data (1 ng to 6 pg gDNA input) were evaluated with a combination of percent-based and static minimum read count AT values. Comparison of varying AT values and the effect on true allele call rates as well as unanticipated read detection was assessed to achieve an ideal balance of data quality and quantity. Additionally, 4 theoretical options for ST application will be offered and include a detailed review of stepwise methods, calculations and resulting data that support a locus “grouping” approach based on locus performance, or intensity groups, within each STR and iSNP marker set.

472 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P391 - ESTIMATION OF EXTRACTION EFFICIENCY BY DROPLET DIGITAL PCR Erica Romsos1, Peter Vallone1

1 National Institute of Standards and Technology, Applied Genetics Group, Gaithersbug, USA

In the literature, extraction efficiency has often been evaluated without the knowledge of the starting quantity of DNA in the beginning of the extraction process. This results in the downstream short tandem repeat (STR) process being the endpoint metric used to determine efficiency of the extraction process on a basis of full and partial genotyping profiles. In this study, extraction efficiency experiments were conducted to evaluate the actual yield of DNA recovered for three extraction methods: manual phenol-chloroform, the Qiagen EZ1 Advanced XL Extraction robot, and the Qiagen QIAamp DNA Mini extraction kit. Three sources of DNA were examined: a human cell line with cells counted via flow cytometry, blood with measured white blood cell count via hemocytometer, and well-characterized extracted DNA quantified with droplet digital polymerase chain reaction (ddPCR). ddPCR allows for the DNA copy number to be determined without the need of an external calibrant. The quantity of each DNA source was varied over a dilution series with replicate extractions for each extraction method. Sample extracts were quantified by in house ddPCR assays targeting autosomal loci [1]. Extraction efficiency was determined by evaluating the amount of sample recovered through extraction to the controlled amount of DNA put into the extraction process for each extraction method and DNA source examined. References 1. Duewer DL, Kline MC, Romsos EL, Toman B. Evaluating Droplet Digital PCR for the quantification of human genomic DNA: Conversion from Copies per Nanoliter to Nanogram Genomic DNA per Microliter. Anal Bioanal Chem (2018) 410:2879. https://doi.org/10.1007/s00216-018- 0982-1.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 473 P392 - EVALUATING THE AMPLIFICATION EFFICIENCY OF MALBAC® SINGLE CELL DNA KIT FOR TRACE DNA (P) Qiannan Xu1,2#, Zhenmin Zhao1#, Jiayi Zhang1, Chengtao Li1, Xiling Liu1

1 Academy of Forensic Science, Shanghai Key Laboratory of Forensic Medicine, Shanghai, China 2 Department of Forensic Medicine, School of Basic Medical Science, Wenzhou Medical University, Higher Education District, Wenzhou, 325035, PR China

DNA typing by using trace DNA is a big challenge in forensic science. To test whether the whole genome amplification technology could resolve this problem, we firstly evaluated the efficiency of whole genome amplification technology for DNA by using the MALBAC® Single Cell DNA Kit to amplify trace DNA and the GoldeneyeTM DNA ID System to genotype STRs. The yield of amplified DNA and the STR typing success rate were then assessed. The DNA yield was found to raise more than 200-fold after the whole genome amplification. Moreover, the STR typing success rate by using the amplified DNA with a template lower than 6.25pg was higher than that without amplification. This study suggested the potential usability of whole genome amplification technology in forensic trace DNA analysis. # Contributed equally * Corresponding authors: Xiling Liu (e-mail: [email protected], phone number: +862052351327) and Chengtao Li (e-mail: [email protected], phone number: +862052351327).

474 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P393 - EVALUATING THE USE OF HYPOXIA SENSITIVE MARKERS FOR BODY FLUID STAIN AGE PREDICTION Graham Williams1, Fisal Asaghiar2

1 Staffordshire University, Department of Criminal Justice and Forensic Science, Stoke-on-Trent, United Kingdom 2 University of Huddersfield, School of Applied Sciences, Huddersfield, United Kingdom

In order to augment DNA profiling and body fluid identification techniques, efforts are being made to increase the amount of information available from a crime scene stain. A key question surrounding crime scene stains is the length of time between the deposition of the stain and its subsequent recovery. For example, is the blood deposited fresh or from an unrelated nose bleed two weeks ago? Or was the semen deposited recently, or on a previous consenting occasion? Once a body fluid leaves the body, the oxygen concentration in the environment changes, therefore it may be that this causes a change in expression of hypoxia-sensitive biomarkers. Here, a range of blood and saliva samples were collected incubation at room temperature for up to 30 days, before undergoing total RNA extraction and cDNA synthesis. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A Gene (VEGFA) and Hypoxia-Inducible Factor 1 Alpha Subunit (HIF1A), with Beta-Actin (ACTB) as a reference gene. A range of linear correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 1.94 days and 4.57 days for saliva and blood respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on stains up to 30 days old with a margin of error ranging from ~2 days through to ~6 days.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 475 P394 - EVALUATION OF FIVE PRESERVATION METHODS FOR RECOVERY OF DNA FROM BONE Sasitaran Iyavoo1,2, Sibte Hadi1, William Goodwin1

1 School of Forensic and Applied Sciences, University of Central Lancashire, Preston, United Kingdom 2 Anglia DNA Services, Scottow Enterprise Park, Norwich, United Kingdom

Preservation of the biological evidences plays paramount importance especially in samples with limited DNA, such as bones and teeth. Effective preservation will enable forensic and medical personnel to confidently store samples until they can be analyzed. Also, in the case of mass disasters, the identification of human remains may be prolonged, thus preservation of those remains to prevent the DNA degradation is very important [1]. The effectiveness of five preservation methods were assessed: cell lysis solution (with 1 % sodium azide), dehydration / freeze drying, ethanol (96 %), freezing and room temperature storage. Preserved bone samples were extracted using five optimized extraction methods [2]. These preservation methods were tested for their efficiency for storage of fresh and degraded bone samples for 6 weeks, 6 months and 1 year. Freezing was found to be the best preservation method for long-term storage of bone samples. This was followed by ethanol (96 %), dehydration/freeze drying and room temperature storage. Full profiles were obtained from bone samples using all these preservation methods. 1. M.D. Gojanovic, D. Sutlovic, Skeletal remains from world war II mass grave: from discovery to identification, Croat. Med. J. 48 (4) (2007) 520–527. 2. S. Iyavoo, S. Hadi, W. Goodwin, Evaluation of five DNA extraction systems for recovery of DNA from bone, Forensic Sci. Int.: Genet. Suppl. Ser. 4 (1) (2013) e174–e175.

476 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P395 - EVALUATION OF RAPIDLY MUTATING Y-STRS ON SOUTH INDIAN PEDIGREE SAMPLES Sasitaran Iyavoo1, Rashed Alghafri2,3, Reem Almheiri2, Thomas Haizel1

1 Anglia DNA Services, Scottow Enterprise Park, Norwich, United Kingdom 2 General Department of Forensic Sciences and Criminology, Dubai Police General Head Quarters, Dubai, United Arab Emirates 3 Biology Department, United Arab Emirates University, Al-Ain, United Arab Emirates

Y-chromosome STR profiling is a useful tool for forensic analysis to process challenging cases such as sexual assault when the male DNA is in small amount compared to the female DNA. However, in the cases where the suspects have the same paternal lineage, conventional Y-STR markers would not be very useful to differentiate them. Thus, Y-STR markers with more discriminative power are required to solve such cases [1]. The effectiveness of rapidly mutating Y-STRs multiplex assay containing 13 RM Y-STR markers [2] was compared with the Yﬁler® kit with conventional Y-STR markers for their efficiency in differentiating males within the same paternal lineage. Buccal swab samples from four generations comprising sixteen South Indian males were analyzed with both systems. More mutations were observed in the RM Y-STRs profiles compared to the Yﬁler® profiles which could differentiate several males within this pedigree samples. This study emphasized the importance of RM Y-STRs when differentiation between males within the same paternal lineage is required. 1. K.N. Ballantyne, V. Keerl, A. Wollstein, Y. Choi, S.B. Zuniga, A. Ralf, M. Vermeulen, P. de Knijff, M. Kayser, A new future of forensic Y-chromosome analysis: Rapidly mutating Y-STRs for differentiating male relatives and paternal lineages, Forensic Sci. Int.: Genet. 6 (2) (2012) 208–218. 2. R. Alghafri, W. Goodwin, A. Ralf, M. Kayser, S. Hadi, A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs, Forensic Sci. Int.: Genet. 17 (2015) 91–98.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 477 P396 - EVALUATION OF SOLVENTS USED TO RECOVER DNA AND RNA FROM CRIME SCENE STAINS Graham Williams1, Fathi Farag2

1 Staffordshire University, Department of Criminal Justice and Forensic Science, Stoke-on-Trent, United Kingdom 2 University of Huddersfield, School of Applied Sciences, Huddersfield, United Kingdom

Many studies have been conducted upon different recovery methods for recovering DNA from items and crime scenes. These have been mainly focussed upon different swabs and swabbing techniques (For example, double swabbing versus single swabbing). However, these studies have provided inconsistent results even where similar analysis methods have been used; suggesting that the critical issue with DNA recovery is not relating to the swab itself. One such possible critical issue is the solvent used to moisten the swabs prior to recovery; whereby the most common moistening solvent by far is sterile/de-ionised water. In addition, no work regarding the recovery of cellular material for RNA analysis has yet been conducted. Here, a range of solvents alongside a range of different recovery methods was used to collect cellular material from different surfaces (Laminated, tile, and cotton) and analysed using qPCR targeting DNA and RNA markers. The solvents evaluated were deionised water, phosphate buffered saline, SDS, lysis buffer, and ethanol. Three common recovery methods were also used (single swab, wet then dry, dry then wet). In all cases, the use of SDS as a moistening agent improved the yield for both RNA and DNA regardless of the swab technique used. De-ionised water performed poorly suggesting that the use of deionised or sterile water should be discouraged as standard practice.

478 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P397 - EVALUATION OF STANDARD SEXUAL ASSAULT EVIDENCE COLLECTION KIT: COMPARISON OF THE SUCCESS RATE OF STR ANALYSIS FROM PSA POSITIVE SWABS VERSUS RINSES Els Jehaes1, Gitte Leijnen1, Eva Nelis1, Werner Jacobs1,2

1 Antwerp University Hospital, Forensic DNA laboratory, Edegem, Belgium 2 University of Antwerp, Department of Forensic Medicine, Antwerp, Belgium

In Belgium, it is a general recommendation to collect both swabs and rinses in sexual assault cases. We evaluated the need of both prelevations for successful DNA analysis based on historical data from January 2003 till March 2015. DNA concentrations and success rate of STR analysis of a total of 213 PSA positive rinses (including 29 anal rinses and 184 vaginal rinses) and 256 PSA positive swabs (including 55 anal swabs and 201 vaginal swabs) were compared. The study includes 154 cases where both a swab and a rinse were analysed simultaneously. Considering all vaginal and anal swabs and all rinses together, the median of the DNA concentration of DNA isolates of swabs is approximately 2.5 times higher than those of the DNA isolates of rinses. Moreover the success rate of obtaining an autosomal STR profile useful to identify the assailant is 72 % for swabs versus 65 % for rinses for the rape cases where both a swab and a rinse were examined simultaneously. In conclusion, swabs were found to have a higher DNA yield and a higher success rate of STR analysis for identifying the alleged assailant than rinses. Eliminating rinses from a sexual assault evidence collection kit means a significant cost reduction and no longer need for cooled preservation. Moreover and important to take into account, gathering the rinse is experienced as inconvenient by the victim and considered not easy to perform by the majority of the forensic physicians.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 479 P398 - EVALUATION OF THE PERFORMANCE OF THE BETA VERSION OF THE FORENSEQ DNA SIGNATURE PREP KIT ON THE MISEQ FGX FORENSIC GENOMICS SYSTEM Magdalena Marcińska1, Maria Wróbel1, Agnieszka Parys-Proszek1, Tomasz Kupiec1

1 Institute of Forensic Research, Forensic Genetics, Kraków, Poland

Massively Parallel Sequencing (MPS), also known as Next Generation Sequencing technologies, are innovative approaches of analysis of genetic markers, supported by the forensic community. The Illumina Beta Version of the ForenSeq DNA Signature Prep Kit is a high-throughput, multiplexing kit allowing simultaneous, high resolution sequencing of 153 identity informative markers (including 27 autosomal STRs, 7 X-chromosomal, 24 Y-chromosomal haplotype markers and 94 SNPs) plus 56 biogeographical ancestry and 22 phenotypic informative SNPs. Imple- mentation of MPS technology in forensic casework laboratories needs comprehensive, internal validation according to SWGDAM guidelines. The aim of this study was to determine with ForenSeq DNA Signature Prep Kit, method’s precision/accuracy, sensitivity as well as possibility of analysis of two persons mixture using DNA with known genetic profiles. Analysis of challenging casework samples coming from various tissues with different stage of decomposition were performed and compared to results obtained with capillary electrophoresis (CE). In order to evaluate the quality and reliability of the acquired results, informative metrics of STR and SNP markers including depth of coverage (DoC), allele coverage ratio (ACR), and sequence coverage ratios (SCRs) were assessed.

480 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P399 - EVALUATION OF THE POWERQUANT® SYSTEM ON THE QUANTSTUDIO™ 5 REAL-TIME PCR SYSTEM Erica Graham1, Jon Drobac1, Jonelle Thompson1, Mary Loten1, Anupama Gopalakrishnan1, Robert McLaren2, Andy Hopwood1, Lotte Downey1, Douglas Storts2

1 Promega Corporation, Genetic Identity, Madison, USA 2 Promega Corporation, Research and Development, Madison, USA

The QuantStudio™ 5 Real-Time PCR System was tested in conjunction with the PowerQuant® System for the quantification and quality assessment of forensic samples. The experiments are based on requirements listed in the Federal Bureau of Investigation (FBI) Quality Assurance Stan- dards for Forensic DNA Testing Laboratories and guidelines outlined by the Scientific Working Group on DNA Analysis Methods. Following an optimization of ramp rate for the QuantStudio™ 5 System, sensitivity, reproducibility, challenging samples (mixtures, degraded and inhibited) studies were performed. The results demonstrate that the precision and dynamic range of the PowerQuant® System on the QuantStudio™ 5 System is consistent with that of the 7500 Re- al-Time PCR System. The ability of the PowerQuant® System to detect mixed, degraded and inhibited samples on the QuantStudio™ 5 System is comparable to that on the 7500 System. Additionally, no sample-to-sample contamination was observed on the QuantStudio™ 5 Sys- tem. Therefore, the QuantStudio™ 5 Real-Time PCR System is a suitable instrument to use in conjunction with the Promega PowerQuant® System to generate predictive, quantitative and qualitative information about forensic DNA extracts.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 481 P400 - EVALUATION OF TWO DNA FORENSIC EXTRACTION METHODS WITH WATER: STERILE VERSUS DISTILLED WATER Beatriz Gómez1, Paula Mármol1, Cláudia Gomes1,2, Ariana Hernández‑Cordero1, Marta Martín‑Arrebola1, Rocío Rosell-Herrera1, Ana María López-Parra1,2, Sara Palomo‑Díez1,2, César López‑Matayoshi1,2, Carlos Baeza-Richer1,2, Eduardo Arroyo‑Pardo1,2

DNA extraction is one of the crucial beginning steps, since any contamination and/or material loss will affect all following procedures. DNA extraction usually follows protocols with standardized reagents, many of which available in quality-controlled commercial kits. Nowadays, there are multiple commercial kits that allow carrying out the extraction rapidly, but often with a high financial cost. With the aim of presenting an alternative to these commercial kits, we propose two new possible DNA extraction methods, based on the presence of different hypotonic solutions (sterile water and distilled water). A total of 117 extractions were carried out over Whatman® FTA® Cards (Sigma-Aldrich®) impreg- nated with blood, using for the extraction two types of water, with the same incubation times, temperature, and number of shakings. For the essays, it was compared the efficiency of using sterile water (for example, water B|Braun, B. Braun Medical Inc.) versus distilled water (Milli-Q, MilliporeSigma®), with two objectives: 1) if the extraction was possible with both types of water, and 2) what kind of water provided the best results. To prove the effectiveness of the extraction method, we proceeded to amplify the genetic material, either nuclear DNA (autosomal STRs and X-InDels), as well as, mitochondrial DNA Hypervariable regions I and II. Our preliminary results showed that DNA extraction was possible with both types of water, but distilled water was the best reagent for the blood extraction, generating higher number of positive results (complete genetic nuclear profiles), and mitochondrial sequences with very good quality (for example, 90 in a scale up to 100). It is our intent to perform in the future a similar extraction process but with water both sterilized and distilled.

482 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P401 - EVALUATION OF USEFULNESS OF FURTHER Y-STR ANALYSIS IN SEXUAL ASSAULT CASES ON PSA POSITIVE SAMPLES RESULTING IN FEMALE AUTOSOMAL STR PROFILING Eva Nelis1, Gitte Leijnen1, Els Jehaes1, Werner Jacobs1,2

1 Antwerp University Hospital, Forensic DNA laboratory, Edegem, Belgium 2 University of Antwerp, Department of Forensic Medicine, Antwerp, Belgium

Samples from male to female sexual assault cases that are positive for the presumptive prostate specific antigen (PSA) test regularly do not result in a male autosomal STR profile. Due to highly unequal proportions of female and male DNA in typical sexual assault samples, routine autosomal STR analysis often fails to detect the DNA of the assailant even after differential extraction of the samples. Previous studies have shown already the value of Y-STR analysis in such cases. In Belgium, forensic DNA laboratories are only allowed to perform Y-STR profiling on sexual assault samples by a specific requisition after routine autosomal STR analysis. However, a commission for additional Y-STR analysis is rather exceptional. In this study, we evaluated the useful of Y-STR analysis. For 100 PSA positive rinses and swabs from male to female sexual assault cases, resulting in female autosomal STR profiling, 17 % resulted in a full or partial Y-STR profile useful for comparison, using the PowerPlex Y23 system (Prome- ga) Y-STR kit. The success rate raised with a higher DNA input in the PCR mix. In conclusion, in sexual assault cases it is useful to perform extra analysis of Y-STRs on the sperm DNA extracts to identify the alleged assailant.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 483 P402 - EVOLUTION OF RAPIDHIT ID SYSTEM Allison Holt1, Cevat Akin1, Charles Troup2, Jacklyn Buscaino2, Mattias Vangbo2, Matthew Ludeman1

1 Thermo Fisher Scientific, Human Identification Business, South San Francisco, USA 2 Thermo Fisher Scientific, Human Identification Business, Pleasanton, USA

Crime fighting in the 21st century necessitates improved investigation methods, and increased partnership between crime labs and investigation units. Rapid DNA technology emerged over a decade ago as a way of confirming a suspect’s identity while still in custody. Improvements in size and usability of recent RAPID DNA technology systems have enabled additional usage in non-laboratory environments such as police booking stations and border checkpoints. The next step in this evolution is the recent enhancements we present here; improved capabilities for use with investigative leads samples. These improved capabilities were achieved by enhancing the PCR amplification, improving lysis and adjusting the analysis parameters to ensure maximum recovery of alleles while still returning 100 % concordant genotypes under these new conditions. The goal of this study was to assess the newly optimized RapidHIT ID System for the analysis of investigative leads samples with Globalfiler Express chemistry. This included low and mid-level saliva and blood samples, mixtures of saliva, purified DNA and casework sample types. Instrument performance was excellent, with all runs completed and 99 % of samples returning a profile. Concordance studies resulted in 100 % concordance for unflagged loci. For sensitivity studies, improved performance with low-level samples was demonstrated. All mixture samples were flagged appropriately. Many of the casework samples returned usable profiles. We show that the new Rapid Intelligence protocol demonstrated a significant improvement in sensitivity. The results of this study conclude that the Applied Biosystems RapidHIT ID System provides a useful solution, allowing law enforcement personnel to generate investigative leads and identify suspects faster.

484 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P403 - EXPLORING OF NEW AGE-RELATED CPGs USING METHBANK DATABASE AND THE PYROSEQUENCING Bowen Xie1, Feng Song1, Mingkun Xie1, Shuangshuang Wang1, Haibo Luo1, Huan Zhao1, Yun Huang1

1 Department of Forensic genetics, West China School of Basic Sciences & Forensic Medicine, Sichuan University, Chengdu, China

Analyzing biological stains in crime scenes for age prediction could be of great significance in searching the suspects and reconstructing the events. DNA methylation, in particular Age-related CpGs (AR-CpGs) as one of the most promising markers for age prediction, was recently studied by various researchers. MethBank is a database that using 24 datasets from 4,577 blood samples to incorporate high-quality DNA methylome maps for human. In this study, we developed a strategy for screening and detecting new AR-CpGs based on both MethBank database and the pyrosequencing methods. Finally, 9 AR-CpGs were selected for DNA methylation analysis. As results, there were 4 novel AR-CpGs resided on 4 gene (LDB2, ACSS3, PRLHR and FJX1) fragments showed remarkable difference between the different age groups. Additionally, 20 blood stains which stored for over one month were detected by pyrosequencing approach. Our results implied that the strategy could be applied as an effective method for AR-CpGs analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 485 P404 - EXTRACTION OF DNA FROM SKELETAL REMAINS BURIED IN ACIDIC SOILS Suni Edson1,2, Ryan Taira3, Gregory Berg3

1 AFMES-AFDIL, Past Accounting Section, Dover AFB, USA 2 Flinders University, College of Science and Engineering, Adelaide, Australia 3 Defense POW/MIA Accounting Agency, Scientific Analysis, Joint Base Pearl Harbor/Hickam, USA

The Armed Forces Medical Examiner System – Armed Forces DNA Identification Laboratory (AF- MES-AFDIL) and the Defense POW/MIA Accounting Agency (DPAA) are partners in the identification of missing United States servicemembers. Skeletal materials are recovered world-wide from past military conflicts; however, this presentation will focus on those recovered from Southeast Asia. The soils in Southeast Asia are known to be acidic, with the majority being classified as acrisol or gleysol (pH 4.4 – 7.2). Acidic soils are thought to compromise the structural integrity of buried skeletal materials, leading to a degradation of the DNA found within. This study evaluates whether this idea is apocryphal or if acidic soils truly have an impact. Samples recovered from Southeast Asia tend to be less successful than those recovered from other conflicts (i.e., World War II and the Korean War) when using Sanger sequencing of mitochondrial DNA (mtDNA). Of the DPAA recovered samples tested in mtDNA between 1990 and 2018, 61 % were successful as compared to World War II (88 %) and Korea (86 %). The success or failure of each tested platform (mtDNA Sanger sequencing, AmpFlSTR®MiniFiler™ and modified AmpFlSTR®Yfiler™) was correlated to the location where the samples were recovered. In addition, each testing strategy was divided by extraction protocol (i.e., organic or inorganic). Samples were plotted against soil maps of the region to determine if there is a large-scale correlation between pH or other soil conditions. Data analysis will provide guidance as to the impact of soil conditions on the quality of skeletal materials and the DNA recovered from within. Extraction protocols designed to remove perceived inhibitors may be overly stringent, resulting in an excessive loss of DNA.

486 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P405 - FACS BASED METHOD FOR EVALUATION OF EFFICIENCY OF THE DNA EXTRACTION METHODS Maarja Sadam1,2, Reet Järving3, Gunnar Tasa3, Marika Väli2

1 Estonian Forensic Science Institute, DNA department, Tartu, Estonia 2 University of Tartu, Institute of Biomedicine and Translational Medicine, Tartu, Estonia 3 Estonian Forensic Science Institute, DNA department, Tallinn, Estonia

Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. Many DNA extraction methods are currently available varying in their ability to efficiently extract the DNA. Most experiments to test DNA extraction methods involve processing replicate tissue samples where the exact number of cells remains unknown. The aim of this study was to develop a cell based model were the number of cells is known and to test its suitability for assessment of efficiency of DNA extraction methods. Peripheral blood mononuclear cells were isolated from venous blood and fluorescence-activated cell sorting (FACS) was used in order to generate samples with controlled number of lympho- cytes. Cells were counted directly on nylon and cotton swabs. DNA was extracted using organic extraction, PrepFiler™ Forensic DNA Extraction Kit (ThermoFisher Scientific) and AutoMag Solu- tion (Ademtech) and quantified using Quantifiler™ Trio DNA Quantification Kit. This novel approach allows us to evaluate efficiency of DNA extraction and also compare effectively different DNA extraction methods. In addition, we show that this model can be used in order to evaluate release yield of different types of swabs.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 487 P406 - FORENSIC ANALYSIS OF MPS mtDNA DATA USING QIAGEN BIOMEDICAL GENOMICS WORKBENCH AND AQME TOOL – PRELIMINARY RESULTS Nair Gouveia1, Pedro Brito1, Benjamin Turner2, Virgínia Lopes1, Ana Margarida Bento1, Filipa Balsa1, Armando Serra1, Lisa Sampaio1, Vanessa Bogas1, Patrícia Cunha1, Marta São Bento1, Maria João Porto1

1 National Institute of Legal Medicine and Forensic Sciences- I.P., Forensic Genetic Service- Centre Branch, Coimbra, Portugal 2 QIAGEN, Bioinformatics, Hilden, Germany

Massively Parallel Sequencing (MPS) technology allows to sequence the entire mitochondrial genome using different mitochondrial DNA (mtDNA) panels. QIAGEN is an example of a company that developed several QIAseq Targeted DNA panels for MPS, including the Human Mito- chondria Panel. These panels have a chemistry based on the incorporation of unique molecular indices (UMIs) before any amplification step, which reduces the number of PCR duplicates and improves the detection of low-frequency variants. However, the analysis of the large amount of mtDNA data produced with MPS may represent a challenge in the forensic area. The Armed Forces Medical Examiner System−Armed Forces DNA Identification Laboratory in collaboration with QIAGEN Bioinformatics designed the AQME tool, customized for forensic analysis of mtD- NA data in the QIAGEN Biomedical Genomics Workbench software, regardless of sample type, library preparation and MPS platform. A set of forensic samples, already sequenced with the Precision ID mtDNA Whole Genome Pan- el (Thermo Fisher Scientific), were performed with the QIAseq Human Mitochondria Panel on the Ion S5™ System, followed by data analysis with the AQME tool in the respective software. In this study, a coverage comparison workflow was created by QIAGEN Bioinformatics, in order to verify the coverage differences between both mtDNA panels for each sample. The preliminary results showed a significant decrease in the Precision ID coverage, after the bioinformatic removal of PCR artifacts. For the QIAseq panel, this coverage variation was lower, since the UMIs enable to know more accurately the initial number of molecules in the original mtDNA sequence.

488 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P407 - FORENSIC APPLICATION OF A mtDNA MINISEQUENCING 52PLEX: TRACING MATERNAL LINEAGES IN SPANISH CIVIL WAR REMAINS Miriam Baeta1, Sandra García-Rey1, Leire Palencia-Madrid1, Caterina Raffone1,2, Marian M. De Pancorbo1

1 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 2 Society of Sciences Aranzadi, Physical Anthropology, Donostia, Spain

Mitochondrial DNA (mtDNA) is the method of choice for tracing maternal lineages from difficult samples in terms of DNA quantity and quality, particularly in identification of mass disaster victims or missing persons, and other complex casework. Currently, the gold standard methodology is still based on sequencing the mtDNA control region, which is technically difficult, time consuming and expensive. Screening methods based on mtDNA SNPs, such as minisiquenc- ing, can be used as an efficient alternative to classify samples according to their haplogroups, and therefore, directly exclude non-matching samples, without the need of a confirmatory full mtDNA sequencing. Here we report the use of a validated minisequencing method, which simultaneously analyzes 52 mitochondrial SNPs, in highly degraded samples. A total of 25 human remains from a mass grave of the Spanish Civil War (1936–1939) were analyzed in order to define their corresponding mtDNA haplogroup and, subsequently, investigate possible maternal relationships with the available reference samples. The results highlight the potential of this minisequencing method as an alternate approach to discriminate among maternal lineages in highly degraded DNA samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 489 P408 - FORENSIC EVALUATION OF 6-DYE CHEMISTRY KIT COMPOSED OF 23 LOCI WITH CASEWORK SAMPLES Naeema Aljanaahi1, Rashed Al Ghafri1, Synan Abu-Qamar2

1 Dubai Police, General Department of Forensic Sciences and Criminology, Dubai, United Arab Emirates 2 United Arab Emirates University, College of Science, Biology Department, Al Ain, United Arab Emirates

The Federal Bureau of Investigation (FBI) has recently expanded the Combined DNA Index System (CODIS) core short tandem repeats (STR) loci from the existing 13 to 20 as new standards, to increase the power of discrimination, reduce the possibility of adventitious matches and expand global data sharing. The majority of casework samples are either low quality, low template or complex mixture, which indeed, requires careful consideration of the derived stochastic variations that lead to heterozygote imbalance, allele dropout and increased detection of background contamination. In this research an internal validation of six-dye STR multiplex assay, Verifiler ™ Plus, is conducted as per the SWGDAM Validation Guidelines for DNA Analysis Methods published in 2016. STR loci comprises 23 autosomal STR loci namely D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, D10S1248, D1S1656, D12S391, D2S1338, D6S1043, Penta D, Penta E), 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel) and Amelogenin (sex-determining marker). The validation study comprises sensitivity, accuracy and precision, stochastic effects, mixtures and contamination assessments. Casework samples will be tested for the ability to improve the efficiency of the assay compared to currently used Globalfiler® assay. Also, the success rate of obtaining positive results from challenging forensic samples that often exist in minimum amount with compromised quality will be evaluated. This research will help decision makers in replacing the currently used DNA profiling system with yet to be released DNA profiling assay Verifiler ™ Plus (Thermofisher).

490 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P409 - FORENSIC IDENTIFICATION AND INTELLIGENCE SNP DATA FROM LATENT DNA USING MASSIVE PARALLEL SEQUENCING Jennifer Young1, Belinda Martin1, Daniel Power2, Piyamas Kanokwongnuwut1, Adrian Linacre1

1 Flinders University, College of Science and Engineering, Adelaide, Australia 2 Thermofisher, Melbourne, Australia

Direct PCR can be used to generate STR profiles from DNA present on the surface of objects. STR profiles are only of use where a potential donor profile is available for comparison and DNA is of sufficient quality and quantity to generate a reliable profile.Often, no donor information is available and only trace DNA is present, therefore more sensitive techniques are required to generate informative genetic data. Massively parallel sequencing (MPS) offers the ability to detect and analyse low quantity and quality DNA present on touched items. Here, we present the first application of direct PCR coupled with MPS to generate both forensic identification and intelligence SNP data from latent DNA. First, we evaluate the QIAGEN 140-SNP forensic identification multiplex sequenced on theIllumina MiSeq. On average, 75/140 SNPs were detected from touch samples which enabled 64/75 samples to be assigned to the correct donor. The number of SNPs detected was not influenced by donor or substrate and a minimum SNP threshold was identified for inclusion of a profile. Secondly, we assess two intelligence-based SNP panels: (1) the HIrisplex System and (2) the Precision ID Ancestry Panel coupled with the automated Ion Chef System and Ion Torrent PGM. Of the 60 touch samples analysed, 54 samples generated phenotype and ancestry predictions. 51/54 samples accurately predicted eye colour and 46/54 predicted correct hair colour. 48/54 samples generated biogeographic ancestry predictions consistent with respective reference samples. However, the confidence of the assignment varied. This study demonstrates the potential of direct PCR with MPS to obtain information from trace levels of DNA, whilst highlights the need for analysis thresholds associated with MPS data.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 491 P410 - GO! DIRECT - FOR FASTER INSIGHTS INTO YOUR FORENSIC CASEWORK SAMPLES Kathrin Wolf1, Mario Scherer1, Anke Prochnow2, Keith Elliott2, Sarah Pakulla-Dickel1, Miroslav Vranes1, Ralf Peist1

1 Qiagen GmbH, Human Identity/ Forensics Product Development, Hilden, Germany 2 Qiagen GmbH, Global Marketing, Hilden, Germany

Many volume crime samples (e.g., from burglaries or vehicle-related crimes) go untested because there is no funding or time available for sample prep. Furthermore, in the US many sexual assault cases lie in storage for months or even years, simply because the effort and complexity of recovering PCR-ready DNA is too great. The newly developed Investigator Casework GO! Kit (QIAGEN) is used for pretreatment of forensic samples in a direct amplification approach. The kit permits the sample lysis, whereas the lysate is compatible with e.g. QIAGEN’s Investigator Quantiplex Pro, and Quantiplex Pro RGQ, or Investigator 24plex QS, ESSplex SE QS or IDplex Plus, without DNA purification. The Investigator Casework GO! Kit can be used for direct amplification of casework samples and thus enables an accelerated workflow in casework analysis. It allows the rapid processing of swabs from casework samples, including cuttings of sexual assault swabs, or other materials, without the need for further purification. The lysate can be used directly for quantification with all Investigator Quantiplex Assays or for DNA profiling using all Investigator STR Assays (unless data from the quantification indicates presence of possible PCR inhibitors). Plus, in sexual assault screening analysis, the Investigator Casework GO! workflow can facilitate the sample processing decision by male-quantification and Auto/Y ratio.

492 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P411 - HIGH THROUGHPUT SEQUENCING DATA ANALYSIS WORKFLOW: mtDNA VARIANT DETECTION AND IDENTIFICATION OF STR/YSTR ALLELES AND ISOALLELES Teresa Snyder-Leiby1, Sarah Copeland2, James Todd3, Wu Jacie4, John McGuigan5, Kayla Hendricks6, Lidong Luo7, C S Jonathan Liu8

1 SoftGenetics- LLC, Forensic Manager, State College, USA 2 SoftGenetics, Forensic DNA Analysis, State College, USA 3 SoftGenetics, Bioinformatics and Programming, State College, USA 4 SoftGenetics, Programming Development, State College, USA 5 SoftGenetics, Bioinformatics, State College, USA 6 Mitotyping Technologies, Forensic DNA Laboratory, State College, USA 7 SoftGenetics, Programming, State College, USA 8 SoftGenetics, Research & Development- VP, State College, USA

High throughput sequencing of mtDNA and STRs enable forensic laboratories to have the benefits of both analysis methods at the same time. HTS chemistries are more cost effective than Sanger sequencing for the mitochondrial genome, produce data at a greater depth of coverage allowing for detection of low level heteroplasmy, and also have the potential to deconvolute mixtures. Advantages of HTS STR chemistries over traditional STR CE include the ability to have smaller amplicons, to analyze more loci in a single reaction, and identification of sequence polymorphisms resulting in isoalleles. Rigorous, user-friendly software is needed in order to analyze the large data files consisting of thousands to millions of reads generated for each sample. GeneMarker®HTS software was released in 2016 as a rapid, user-friendly, software for forensic mtDNA high throughput sequencing data analysis. Development of autosomal and Y-STR capabilities started in 2017; providing genotype and SNP detection within amplicons. National Institute of Standards and Technology (NIST), in conjunction with Promega corporation, gener- ously supplied fastq sequence files and corresponding CE allele calls for 672 samples amplified with the PowerSeq® Auto/Y System and analyzed on an Illumina® MiSeq. Results of these data analyzed in GeneMarkerHTS software were highly concordant with the CE allele calls. Summa- ry of the allele calls concordance and examples of instances where alleles exhibited sequence variation will be presented. Additionally, a review of the mtDNA genome forensic alignment, heteroplasmy report, import of major variant report to EMPOP, sample comparison reporting, and database options will be presented.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 493 P412 - HISTOLOGICAL SPECIMENS IN GENETIC IDENTIFICATION OF NN CADAVERS AND PATERNITY TESTING Magdalena Konarzewska1, Zofia Jagielska1, Tadeusz Malewski2, Witold Pepiński3, Ireneusz Soltyszewski4

1 Medical University of Warsaw, Department Forensic Medicine, Warsaw, Poland 2 Museum and Institute of Zoology- Polish Academy of Sciences, Department of Molecular and Biometric Techniques, Warsaw, Poland 3 Medical University of Bialystok, Department of Forensic Medicine, Bialystok, Poland 4 Warmia and Mazury University in Olsztyn, Department of Criminalistics and Forensic Medicine, Olsztyn, Poland

Collection of biological samples is not a routine practice at each autopsy. The collection may be omitted on purpose (e.g. identification of the corpse is complete or preliminary) or neglected by the pathologist. Tissue samples for pathological examination are commonly excised at the autopsy and frequently offer the only biological material available for DNA analysis. The objective of this study was evaluation of paraffin tissue specimens as a potential source of DNA templates for kinship analysis, depending on a tissue type and a fixation method. The study material comprised 10 sets of five paraffin-embedded specimens of cadaver tissue (a total of 50 samples) with no signs of decomposition – an archival material of the Department of Forensic Medicine, Medical University of Warsaw: three sets dating 2001 and three sets dating 2010 - fixed with 10 % formaldehyde, four sets dating 2018 - fixed with buffered formaldehyde. DNA was extracted using Maxwell® 16 Forensic Instrument (Promega). PowerQuant System (Promega) 7500 Real-Time PCR System (Applied Biosystem) were used for DNA quantitation and integrity assessment. DNA templates were amplified with use of PowerPlex Fusion6C (Promega) and genotyped in 3500XL Genetic Analyzer. Based on our data, DNA degradation in 10 % formaldehyde-fixed tissues increased, while the specimens fixed with buffered formaldehyde yielded good quality DNA. Based on DNA quantitation results and saturation of the PowerPlex Fusion6C profiles, the lung tissue and the brain tissue were the best sources of DNA templates out of all the specimens investigated regardless of the fixing solution used.

494 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P413 - HOW MANY STR MARKERS ARE ENOUGH? Antonella Belen Penacino1, Linda Elsztein2, Mariano Estrada2, Gustavo Penacino1

1 Official College of Pharmacists and Biochemists and INGEN Foundation, DNA Analysis Unit, Buenos Aires, Argentina 2 Official College of Pharmacists and Biochemists, DNA Analysis Unit, Buenos Aires, Argentina

The knowledge of relatedness between individuals is central to many studies in population genetics. To obtain estimates of relatedness with sufficient accuracy, the number of polymorphic markers and the number of alleles per marker is a crucial factor. The aim of our study was to examine the ability of 15 and 22 STR markers included in the Powerplex16 and Powerplex Fusion Kits (Promega Corp) to detect biological kinship, even in presence of presumtive mutations. The data were taken from 200 paternity cases, trios and duos. Each trio consisted of mother, one child and one alleged father, whereas each duo consisted of the same individuals without mother´s data. In addition, we analyzed 100 non-paternity cases (half-siblings, full siblings, uncle-niece, grandfatherhood) and we used the Familias program (Egeland, Thore et al) to calculate all probabilities and likelihood ratios (LRs). We graphed quantity of cases vs LR logarithm, and established a LR upper than 10000 to consider our conclusion as certain. The results have shown that all the trio cases could be solved in both situations (15 or 22 STR markers), while in presence of one mutation only were solved all cases using 22 STR markers, as occured with the duos. Faced with the presumtion of two mutations, none of the results using 15 STRs were conclusive. Considering that around of 90 % of cases analyzed in our laboratories are paternity cases (duos and trios), would be more useful to develop a system with markers completely differents to all we use, instead the commercial kits with some more STRs. This alternative system would be used only in complex cases (two mutation or deficiency cases), when no conclusive results were obtained using the 22 common STRs.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 495 P414 - IDEAL STR KIT FOR DIRECT PCR ON TOUCH DNA SAMPLES Belinda Martin1, Adrian Linacre1

1 Flinders University, College of Science and Engineering, Bedford Park, Australia

We report on the comparison of six routinely used STR kits to further our understanding into the ideal analysis of touch DNA samples by direct PCR. This study provides laboratories with data to support their STR kit choice in any research or analytical work relating to touch DNA by direct PCR. The STR kits tested were as follows: GlobalFiler®, Identifiler® Plus, Identifiler® Direct, VeriFiler™ Plus, from Thermo Fisher Scientific; PowerPlex® 21 from Promega Corporation; and the Investigator 24plex QS kit from QIAGEN. Eight donors washed their hands and, after 15 m, touched a range of items, according to regular use, for up to 15 s. The items comprised: an insulated wire, a zip- lock bag, a piece of circuit board, a matchstick, and a fingermark on glass. All substrates were cleaned before use and negative controls were collected. All testing was performed in triplicate with a total data set of 120 per kit (or 960 including all donors). The items were double-swabbed using an ultra-fine nylon swab pre-moistened with 0.1 % Triton™ X-100. The tips of the swabs were placed directly into a 0.2 mL PCR tube and were amplified following the manufacturers’ recommended. Average allele amplification success, baseline noise and artefact presence differed between kits, i.e. Identifiler® Plus generated more profile alleles on average than GlobalFiler®. We will present discussion as to the quality of the data generated from each kit and the ease of interpretation. The data generated allows laboratories to optimise their workflow, through informed kit selection, when implementing direct PCR to ensure the highest DNA profiling success. The results also further illustrate the advantages of direct PCR from touch DNA as near complete DNA profiles were generated from items touched for only 15 s.

496 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P415 - IDENTIPLEX: A NEW STR KIT FOR HUMAN IDENTIFICATION DEVELOPED AND VALIDATED AT IDENTIGEN LAB (COLOMBIA) German Burgos Figueroa1, Juan David Granda2, Oscar Palacio2, Carmen Giselle Freyle-Barón2, Kelly Michelle Maldonado-Uquillas3, Adriana Ibarra2

1 Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 IdentiGEN - Genetic Identification Laboratory and Research Group of Genetic Identification, Institute of Biology, School of Natural and Exact Sciences FCEN, University of Antioquia, Medellin, Colombia 3 Carrera de Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas UDLA, Quito, Ecuador

Colombian laws require that paternity testing probability (W) values be higher than 99,99 % to prove biological relationship. If this value is not reached, the test must be declared NON-CON- CLUSIVE. IdentiGEN lab currently uses 23 autosomal genetic markers included in the commercial kits Identifiler®, PowerPlex®16, FFFL® and Triplex® (in-house kit: D18S535, D2S1338, SE33); however, in some complex cases the number of markers is not enought to reach the legal statistical threshold. In the present study an in-house kit named IdentiPLEX, consisting of 19 markers (D5S818, D13S317, D7S820, D16S539, PENTAE, D2S441, VWA, D12S391, CSF1PO, PENTAD, D1S1656, TH01, SE33, D10S1248, F13B, D21S11, D18S51, D22S1045 and Amelogenin) was developed. The aforementioned loci were selected from NIST & STRbase databases. The kit was validated according to the requirements established by the norm NTC-ISO17025, the international and Colombian recommendations for forensic genetics labs; these include among others, studies of precision, specificity, sensitivity, stutter proportion and concordance tests. Forensic and population parameters were calculated using Powerstats and Arlequin V3.5.2 softwares with data from 701 individuals previously typed as described. The IdentiPLEX kit generates accurate genetic profiles, reproducible and concordant with comparative commercial kits and demonstrated to be human specific. Furthermore, all markers included in the IdentiPLEX kit were found to be in Hardy-Weinberg equilibrium and did not present linkage disequilibrium. The previous analyses allowed us to reaffirm that the loci studied are appropriate for routine analysis casework, and more specifically in the solution of complex cases, where it helped to solve 6 from 21 reanalyzed (28.57 %).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 497 P416 - IDENTITY SNP TYPING VIA THE FORENSEQ ASSAY: IMPLICATIONS FOR PRACTICAL USE Rebecca Just1, Jennifer Le1, Michelle Galusha1, Lilliana Moreno1

1 FBI Laboratory, DNA Support Unit, Quantico, USA

Data from internal validation studies of the ForenSeq system (Verogen, Inc.) using DNA Primer Mix B (DPMB) were used to develop analytical, stochastic and intralocus balance thresholds for the 92 identity-informative SNPs (iSNPs), and to obtain a baseline understanding of interlocus balance across the marker set. Under the experimental conditions, notable findings included a very low incidence of non-authentic alleles above the ForenSeq Universal Analysis Software (UAS) detection threshold, which should enable use of a low analytical threshold and thereby increase the utility of the assay. Testing of the determined thresholds against 15+ further assay runs 1) confirmed the use of a percentage-based analytical threshold and a fixed read count stochastic threshold, 2) suggested that use of different stochastic thresholds for different starting DNA inputs could help to maximize allele recovery, and 3) revealed variations in marker performance across DPMB lots. This presentation will discuss implications of these findings for practical use of the ForenSeq system for iSNP typing, including threshold setting within the UAS to balance data recovery and data analysis workload, expectations for locus and allele recovery, and internal quality control of the assay.

498 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P417 - IMPACT OF DNA DEGRADATION ON MASSIVELY PARALLEL SEQUENCING-BASED AUTOSOMAL STR AND SNP NUCLEAR DNA TYPING SYSTEMS Elena Zavala1, Swetha Rajagopal1, George H. Perry2, Ivana Kruzic3, Bašić Željana3, Thomas Parsons4, Mitchell M. Holland1

1 The Pennsylvania State University, Biochemistry and Molecular Biology- Forensic Science Program, University Park, USA 2 The Pennsylvania State University, Anthropology and Biology, University Park, USA 3 University of Split, Forensic Science, Split, Croatia 4 International Commission on Missing Persons, Science and Technology, The Hague, Netherlands

The degraded nature of DNA recovered from older skeletal samples makes it difficult to use short tandem repeat (STR) analysis, which targets DNA segments ranging from 80 to 500 base pairs (bp) in length. Analysis of single nucleotide polymorphism (SNP) loci with recently developed STR assays using a massively parallel sequencing approach may serve as a solution to the problem, as they can be typed by targeting templates down to 60 to 170 bps. We performed a modeling study using the ForenSeq kit (Verogen, San Diego, CA) that demonstrates that SNPs can increase the significance of a human identification when analyzing highly fragmented DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. This data was also used to evaluate the current default thresholds for the Universal Analysis System (UAS) in relation to degraded samples. Resulting observations of stutter, allelic dropout and heterozygous balance will be discussed. A comparison between this modeling study and results from human skeletal material (n=14, 9th to 18th centuries) further demonstrated the utility of the ForenSeq kit for highly degraded samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 499 P418 - IMPACT OF MUTATION IN A LOCUS D2S441 ON ELECTROPHORETIC MOBILITY Maarja Sadam1,2, Reet Järving3, Gunnar Tasa3, Marika Väli2

There are a variety of commercial STR multiplex kits with different configurations of STR markers and different primer sequences available to the forensic community. Concordance between different STR kits used for DNA profiling is very important in domestic comparison of DNA profiles and also one of the crucial elements for international DNA data exchange. Therefore, concordance is extensively studied by the companies during developmental validation as well as by individual laboratories during in-house validation. Here we describe a case where the same amplicon resulted in different genotypes on genetic analyzers 3130 and 3500xL. Moreover, the use of different kits on the same genetic analyzer, gave different heterozygous genotypes. The sequence information of the affected alleles revealed mutation in a flanking region of D2S441 locus. Analysis with nucleic acid folding and hybridization prediction software showed that the mutation leads to profound changes in a secondary structure of the DNA. We suggest that altered secondary structure influences the electrophoretic mobility of the DNA fragment, which ultimately results in discordant DNA profiling results using different STR kits and different genetic analyzers.

500 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P419 - IMPLEMENTATION OF PREP-N-GO™ BUFFER FOR DNA EXTRACTION FROM BUCCAL SWABS Sasitaran Iyavoo1, Simon Knights1, Michalis Mavrommatis1, Thomas Haizel1

1 Anglia DNA Services, Scottow Enterprise Park, Norwich, United Kingdom

DNA extraction from buccal swabs is very reliable considering the DNA yield, rapidness and also the non-invasive nature of sample collection [1]. Anglia DNA Services processes buccal swabs for the paternity and relationship testing using the QIAamp® DNA Minikit. DNA extraction using this kit takes approximately 2 hours for a batch of samples and involves several tube transfers. To reduce the turnaround time and avoid any cross contamination or samples mix-up, a simpler and easier DNA extraction method; Prep-n-Go™ Buffer was tested. Two methods were recommended by the Prep-n-Go™ Buffer manufacturer for buccal swab extraction; room temperature and heat protocols [2]. Two buccal swabs from each individual taken part in this study were collected from thirty anonymous volunteers. One swab was extracted using the room temperature protocol while another one with the heat protocol for comparison study. Extracted DNA were amplified directly using the VeriFiler™ Express PCR Amplification kit. Room temperature protocol produced good quality DNA profiles while the heat protocol produced stronger DNA profiles with artefacts such as pull-ups and split peaks. Statistical analysis on peak heights showed a significant difference between both protocols. Based on this study, room temperature protocol was implemented for buccal swabs DNA extraction at Anglia DNA Services. With this new method which only involves a single tube, DNA extraction can be done in less than an hour for a batch of samples. 1. C.A. Ruiz, M.E. Chaney, A.J. Tosi, Medical-grade buccal swabs versus drugstore cotton swabs: No difference in DNA yield, MethodsX 5 (2018) 39–42. 2. VeriFiler™ Express PCR Amplification Kit User Guide, ThermoFisher™ Scientiﬁc, 2017.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 501 P420 - IMPLEMENTING AN OPTIMIZED DNA EXTRACTION PROTOCOL FOR ANCIENT AND HIGHLY DEGRADED SAMPLES Catarina Xavier1, Mayra Eduardoff1, Barbara Bertoglio2,3, Andrea Casas-Vargas4, Walther Parson1,5

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Department of Public Health, Experimental and Forensic Medicine, Section of Legal Medicine and Forensic Sciences, University of Pavia, Pavia, Italy 3 LABANOF- Laboratory of Forensic Anthropology and Odontology, Department of Biomedical Sciences for Health, Section of Legal Medicine, University of Milan, Milan, Italy 4 Grupo de Genética de Poblaciones e Identificación, Instituto de Genética, Universidad Nacional de Colombia, Bogotá, Colombia 5 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

DNA extraction is a crucial step in the forensic routine workflow and more challenging in the processing of human solid remains such as bone and teeth, due to the common degraded state of these biological materials. By determining the DNA quality and quantity in downstream forensic analysis, the development and optimization of new DNA extraction methods is extremely relevant for the forensic genetics community. A new extraction protocol was adapted from an ancient DNA research study [1], implemented and optimized in a forensic DNA processing laboratory. Systematic alterations in the protocol have been made to allow for a full optimization of the method, such as incubation time and temperature and different extraction and binding buffer combinations. In addition, different amounts of grinded bone/tooth were tested to assess the effect of full bone/tooth powder dissolution in extraction buffer on DNA recovery. Finally, a thorough comparison was performed between both old lab-established and the new method on DNA extraction efficacy by real-time qPCR quantification of the extracts stemming from remains of different origins and ages (30, 800–1500 and 2000 years). The new extraction method brings the advantage of using a small amount of grinded bone or tooth and a shorter, more streamlined protocol, thus being a valuable contribution to the entire forensic genetics field and especially to laboratories currently dealing with old/highly degraded remains. [1] Dabney, J., et al., Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. PNAS, 2013. 110(39): p. 15758.

502 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P421 - IMPORTANCE OF DNA ANALYSIS FOR IDENTIFICATION AND CONFIRMATION OF HUMAN REMAINS, FOLLOWING A FORENSIC AUTOPSY Dragana Zgonjanin1, Eida Almohammed2, Dalibor Nedić3, Aleksandra Milić4, Stojan Petković1

1 Clinical Center of Vojvodina, Institute of Forensic Medicine, Novi Sad, Serbia 2 Ministry of Interior of Qatar, Qatar Forensic laboratory, Doha, Qatar 3 Clinical Center of Banja Luka, Institute of Forensic Medicine of Republic of Srpska, Banja Luka, Bosnia and Herzegovina 4 University of Novi Sad, Faculty of Medicine, Novi Sad, Serbia

Various forensic techniques are used to identify a human corpse, depending on the circumstances and the state of remains. Unfortunately, the standard forensic identification methods were not sufficient in 30–35 % of all victim, therefore DNA identification was necessary. We have found that forensic autopsy does not always give reliable answers to important questions: What is the time laps between the moment of death and the skeletal remains discovery? What is the age of the deceased? In fact, it could often cause an erroneous identification strategy choice. This paper describe forensic application of current DNA technology to solve a missing person’s case. The disappearance of 57 year old male was reported in a town in the north of Serbia in Au- gust 2017. In January 2018, in that geographical area parts of skeletal remains, remains of cloths and a watch were found and sent to the Institute for Forensic Medicine. The age of the person was estimated to be between 75–80 years, and it was estimated that the remains had been buried for more than 10 years. This report confused the police, because they did not have a missing person of the above description on the record. However, the DNA analysis of a bone sample has shown that the remains belong to the 57 year old missing person who disappeared 5 months prior and for whom the son was the reference sample donor. Complete DNA profiles obtained using AmpFℓSTR® Identifiler® Plus, AmpFℓSTR® NGM™, GlobalFiler™ and AmpFℓSTR® Yfiler® kits were a match with the reference sample of victim’s son (probability 99.9995147 %). This work has shown that for identification of skeletal remains and solving missing persons cases, the forensic application of the latest DNA technology is of utmost importance.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 503 P422 - INITIAL ASSESSMENT OF NGS AS A TOOL TO AUGMENT ROUTINE CE ANALYSIS OF STRs Andrew Revoir1, Holly Lancaster1, Carole Ames1

1 Metropolitan Police Service, Forensic Services, London, United Kingdom

The introduction of Next Generation Sequencing (NGS) into forensic casework requires a detailed appraisal of how the technology can assist with improving current CE based methods bearing in mind the investment required to implement this significantly different method into operational forensic laboratories. To this end an initial assessment was conducted which involved the typing of 53 samples using the Verogen ForenSeq™ DNA Signature Prep Kit in conjunction with the Illumina MiSeq FGx™ DNA Sequencer. Concordance was checked for Autosomal and Y-STRs present in both the Verogen kit and the Promega PowerPlex® Fusion 6C kit when typed by allele size alone. In addition all alleles typed were analysed for sequence structure with NGS versus size calling with CE in order to ascertain the likely benefit of analysing STRs by sequence as opposed to size alone.

504 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P423 - INTERNAL VALIDATION OF LATEST GENERATION STR KITS FOR DIRECT STR TYPING FROM REFERENCE SAMPLES Phuvadol Thanakiatkrai1, Wannarat Nongmanee2, Pirunpat Aitthiprajak2, Sukanya Phetpeng2, Auttapol Viset2, Arjaree Ujchariyakajorn2, Nuryanee Sabahanaloh2, Sakawrat Aoumtas2, Thitika Kitpipit1

1 Prince of Songkla University, Department of Applied Science, Hat Yai, Thailand 2 Office of Police Forensic Science Center 10- Royal Thai Police, Biology and DNA Sub-Division, Yala, Thailand

Forensic STR kits are manufactured by various biotechnology companies. Differences in primer designs and proprietary chemicals, as well as the availability of instruments in each laboratory, may result in performance differences (e.g. first-pass success rate, inhibitor tolerance, interlocus balance, and null alleles). As such, internal validation is a necessary step prior to adopting a new kit, especially if accreditation is sought. Here, we evaluated three latest generation direct STR kits of three manufacturers (Thermo Fisher Scientific’s GlobalFiler® Express – 24 loci, Prome- ga’s PowerPlex® Fusion 6C – 27 loci, and Qiagen’s Investigator® 24plex GO! – 24 loci) for direct STR typing of reference samples as part of ISO17025:2017 implementation. Thirty buccal swab samples (untreated cotton swabs) were used to optimize the parameters that could affect test results, such as the amount of swab used, PCR cycle number, and electrophoretic injection condition. Each kit was then used for direct STR typing of additional one hundred untreated buccal swabs. Analysis was done using the 3500xL Genetic Analyzer with GeneMapper™ ID-XSoftware. A scoring system based on profile quality was used to evaluate the kits: high-quality, minor-issue, moderate-issue, and critical-issue. GlobalFiler® Express kit showed the highest number of high-quality profiles (i.e. full profile with no quality flag), followed by PowerPlex® Fusion 6C and Investigator® 24plex GO!. Non-standard loci typed in the kits (e.g. DYS391 and “Quality Sensors”) provided additional information in cases where amelogenin-Y failed to amplify and also where inhibitors were present. In summary, we selected the GlobalFiler® Express kit for direct STR typing of untreated buccal swab samples due to its superior performance.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 505 P424 - INTERNAL VALIDATION OF SureID 21G HUMAN STR IDENTIFICATION KIT (HEALTH GENE TECHNOLOGIES) Anna Barbaro1, Patrizia Cormaci1, Angelo La Marca1

1 Studio Indagini Mediche E Forensi SIMEF, Forensic Genetics, Reggio Calabria, Italy

The SureID® 21G Human STR Identification kit™, produced by Health Gene Technologies, uses a fast PCR fast-cycling PCR technology, which allows in a single multiplex assay, in less than 2 hours, the amplification of 20 autosomal STR loci and the sex locus Amelogenin, using a 5-dye technology. This kit is designed for forensic applications and paternity cases. In the present study we performed the kit internal validation, evaluating its efficiency either on control samples than on a wide range of forensic samples when using reduced reaction volumes or direct amplification. Some critical parameters such as sensitivity, precision, reproducibility, stochastic effects, intra/ intercolor balance, peaks balance ratio, mixture detection have been studied. Results demonstrates the use of reduced reaction volume, without altering components ratio, does not affect the SureID® 21G kit performance, allowing reliable and efficient analysis also of challenging casework samples. In addition SureID® 21G kit may be successfully used for the direct amplification of a wide range of forensic samples without any need for extraction or purification : this allows reducing procedure time and costs.

506 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P425 - INVESTIGATING THE RESOLUTION OF ANCESTRY TESTING IN GEOGRAPHIC REGIONS CHARACTERIZED BY A HIGH POPULATION ADMIXTURE Farida Alshamali1, Biduth Kundu2, Luisa Pereira3

1 Dubai Police GHQ, Gen.Dept. Forensic Science &Criminology, Dubai, United Arab Emirates 2 Integrated Gulf Biosystems- Dubai- UAE, Dubai, United Arab Emirates 3 Ipatimup Instituto de Patologia e Imunologia Molecular da Universidade do Porto- Porto- Portugal and i3S Instituto de Investigação e Inovação ao em Saúde- Universidade do Porto- Porto- Portugal, Genetic Diversity Group, Porto, Portugal

We have been conducting several genomic studies across the Arabian Peninsula, confirming it is a melting pot of African, European and Asian. The admixture has been taking place since pre-historic times, and attained an astounding dimension at contemporary times in places such as the UAE. This phenomenon challenges the use of ancestry testing in forensic genetics, which could provide additional information on a suspect or strengthen eyewitness accounts. It is thus important to evaluate the level of resolution these tests provide when applied in the context of migration nexus such as the UAE. We performed the genotyping of the Applied BiosystemsTM Precision ID Ancestry Panel in 30 residents in Dubai, UAE, born in different countries: 21 UAE, 2 Yemeni, 1 Saudi Arabian, 1 Omani, 1 Iranian, 1 Pakistani, 1 Ethiopian, 1 Sudanese and 1 Egyptian. Ancestry was inferred with the HID SNP Genotyper™ plug-in. Only 3 Arabians showed a 100 % Southwest Asian profile, 16 of them showed a main Southwest Asian (SWA) proportion (between 40 % and 90 %) admixed with one or two other ancestries, while the remaining 6 were highly admixed. Even so, all the Arabian individuals were mapped around SWA. The Egyptian profile resembled a low admixed Arabian (95 % SWA), while the Iranian had European (60 %) added by SWA (30 %) components. The Pa- kistani had the highest South Asian profile (70 %), mixed with three other ancestries. The 2 Af- ricans were also admixed (Ethiopian – 45 % African and 55 % SWA; Sudanese – 30 % and 70 %, respectively). This work shows that the application of ancestry tests can be informative in helping to solve forensic caseworks by providing information for investigative leads, but values should not be strictly interpreted given the highly admixed profile of some populations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 507 P426 - INVESTIGATION OF THE EFFICIENCY OF WHOLE GENOME AMPLIFICATION PRIOR TO SHORT TANDEM REPEAT ANALYIS USING DEGRADED DNA Mitsuyo Machida1, Kazuhiko Kibayashi1

1 Tokyo Women’s Medical University, Department of Legal Medicine School of Medicine, Tokyo, Japan

Short tandem repeat (STR) analysis is prone to failure since DNA is frequently degraded by various environmental factors. An increase of the number of starting templates may improve the success of STR profiling. One approach to increase the number of DNA templates is whole genome amplification (WGA), however, few studies have shown the ability to evaluate the availability of WGA for degraded DNA samples in forensics. Therefore, we performed PCR-based WGA called “modified improved primer extension preamplification (mIPEP)” prior to STR analysis using degraded DNA, since the PCR-based WGA method is less affected by DNA quantity and quality. Saliva from 4 volunteers was previously dried onto filter papers. These samples were irradiated to UVA light (365 nm) for 3, 7, 14, and 30 days. The mIPEP method was initiated using 5, 0.5, and 0.05 ng of DNA after DNA extraction. Following the mIPEP, STR analysis was performed using the AmpFlSTR Identifiler PCR Amplification Kit. This study was approved by the ethics committee at Tokyo Women’s Medical University. The number of detectable STR loci with mIPEP was smaller than that without mIPEP using 5 and 0.5 ng of DNA in untreated samples. After 3 days of irradiation, the number of detectable STR loci with mIPEP was smaller than that without mIPEP using 0.5 and 0.05 ng of DNA. There was no significant difference between the number of detectable STR loci without and with mIPEP after 7 and 14 days of irradiation. However, the number of detectable STR loci with mIPEP was greater than that without mIPEP using 5 and 0.05 ng of DNA after 30 days of irradiation. Hence, mIPEP was not necessary for undegraded to moderately degraded samples, however, mIPEP was effective using severely degraded samples to improve the success of STR profiling.

508 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P427 - IS GENOMIC DNA EXTRACTED AND STORED AT -20 °C FOR LONG TIME USEFUL IN FORENSIC FIELD? Giulia Sguazzi1,2, Flavia Lovisolo3, Sarah Gino4,5

1 University of Piemonte Orientale, Department of Health Sciences - Biotechnology, Novara- via Solaroli 17- 28100, Italy 2 University of Pavia, Department of Biology and Biotechnology “L.Spallanzani” - Experimental and Applied Biology, Pavia- 27100, Italy 3 University of Piemonte Orientale, Department of Health Sciences, Novara- via Solaroli 17- 28100, Italy 4 University of Torino, Department of Public Health and Pediatrics - Laboratory of Criminalistic Sciences “Carlo Torre”, Torino- Corso Galileo Galilei 22- 10126, Italy 5 University of Piemonte Orientale, Department of Health Sciences, Novara- via Solaroli17- 28100, Italy

With the progress of technologies, forensic genetic laboratories have been more frequently involved in “cold case”. The aim of this study was to understand whether the DNA extracted and stored for a long time at-20°C could be useful for new analyses to identify the perpetrator of unsolved crimes, especially when evidence is not longer available.We selected 120 DNA samples obtained from evidence collected at the crime scene between 2001 and 2010: they were quantified again using Realtime PCR and the “Plexor HY System” kit.The results have been compared with those obtained in the past. For 37 samples discrepancies were observed (i.e. positive quantification, identification of male material mixed with female ones). Then these samples were amplified with “AmpFISTR Identifiler Plus” and “PowerPlex ESI 17 Fast System” kits. Genetic profiles useful for a comparison were obtained for all the 37 analysed samples. In 5 samples mixed profiles were highlighted, unlike what was obtained in the past, and in addition, in 28 cases, where no genetic profile was previously typed, a genetic profile was also identified.The study shows how it is possible, even after a considerable time interval, to still obtained genetic profiles useful for a comparison, as well as the possibility of typing new ones. Comparing the results of the analysis of DNA polymorphisms it is possible to underline the improvement that the current techniques have brought to the typing of genetic profiles: regarding the attribution of gender and the perception of contributors within mixtures.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 509 P428 - LARGE-SCALE CONCORDANCE STUDY FOR 16 AUTOSOMAL STRs ANALYSED WITH PowerPlex ESI 17 PRO AND NGMSelect Alexandra Haas1, Silvia Utz1, Martin Zieger1

1 Institute of Forensic Medicine, University of Bern, Forensic Molecular Biology, Bern, Switzerland

The concordance of STR typing results obtained with different kits is extremely important when comparing STR profiles from different labs and is therefore crucial for keeping national databases as error-free as possible. However, when using kits with different sets of primers to amplify the same markers, discordances of STR typing results cannot be excluded. It is therefore of great importance to gain a thorough understanding of the frequency and different types of discordances in order to adjust the search parameters of databases accordingly and to prevent potential matches from going undetected. In our lab, the PowerPlex ESI17 Pro and NGMSelect kits are routinely used for buccal swab analysis. The fact that both of these kits cover the same 16 markers allowed us to identify kit-specific variations and conduct a long-term concordance study. Over a period of 6 years, we identified and documented the occurrence of kit-specific discordances and genetic particularities in a total of more than 25’000 routine samples typed with the PowerPlex ESI17 Pro and NGMSelect kits. The most common events detected were kit-dependent dropouts, the majority of which occurred in profiles obtained with the NGMSelect kit. Dropouts were found to occur most frequently in markers SE33 and vWA. In addition to dropouts, we also documented and quantified the occurrence of tri-allelic patterns, peak imbalances and discordant allele calls. In marker SE33, a repeatedly occurring variant allele was observed when analysed with PowerPlex ESI17 Pro and a small number of events, such as artifact peaks, peak shifts and additional off-ladder peaks, could not be sorted into one of the abovementioned groups.

510 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P429 - LOST AT SEA: A PILOT STUDY INVESTIGATING DNA RECOVERY FROM TEETH IN A SOUTH AFRICAN NATURAL MARINE ENVIRONMENT Chandra Finaughty1,2, Victoria E. Gibbon2, Belinda Speed1,2,3, Laura Heathfield1

1 University of Cape Town, Department of Pathology, Cape Town, South Africa 2 University of Cape Town, Department of Human Biology, Cape Town, South Africa 3 University of Cape Town, Department of Oceanography, Cape Town, South Africa

Extracting DNA for human identification from marine wash-ups recovered after long periods is challenging. Research investigating DNA recovery from samples exposed to seawater is also lacking and limited to artificial or semi-realistic environments. South Africa poses a unique context with the high number of unidentified remains and marine wash-up cases along its long coastline. The aim of this pilot study was to assess DNA recovery from pig (Sus scrofa) teeth (n=28) submerged in-situ in a natural marine environment of South Africa. Pig origin was confirmed by DNA barcoding. DNA recovery was assessed by quantitative real time PCR, where amplification of nuclear DNA was successful in 60 % (17/28) of samples for a 96 bp fragment, and in 46 % (13/28) for 200 bp fragment. By comparison, mitochondrial DNA was detected in 57 % (16/28) for a 486 bp fragment. Although in very low quantities, DNA was consistently amplified in teeth submerged during winter, even at 56 days. Suggesting colder seawater temperatures contributed towards DNA preservation. Overall, DNA quantities were comparatively lower than what has been reported for artificial seawater environments. Lastly, non-specific amplification was present within 11 % (3/28) of samples. Further investigation by sequencing suggested it came from a marine microbe species, Oceanisphaera. It is hypothesised that the presence of marine bacteria contributes towards DNA degradation. This study was the first of its kind conducted in a South African marine environment. It demonstrates that DNA recovery from teeth submerged in a natural marine environment is complex; supporting that artificial environments overestimate DNA yield. Future studies on human samples is required to improve success rates of identifying marine wash-ups.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 511 P430 - MASSIVELY PARALLEL SEQUENCING (MPS) TECHNOLOGIES APPLIED TO IMPROVE THE RESOLUTION OF FORENSIC CASEWORK Ana Mosquera Miguel1, María de la Puente1, Ana Freire-Aradas1,Manuel Fondevila Alvarez1, Adrián Ambroa1, Jorge Ruiz-Ramirez1, Lorena Girón-Santamaría1, Ángel Carracedo1, Christopher Phillips1, María Victoria Lareu Huidobro1

1 Institute of Forensic Science, University of Santiago de Compostela, Forensic Genetic Unit, Santiago de Compostela, Spain

Massively Parallel Sequencing (MPS) technologies have emerged in the last few years offering a range of forensic assays to extend the current genetic information obtained with routine forensic DNA analysis. Key advantages of MPS technologies include: much increased information captured as sequence data from different marker types (and their flanking regions); capacity to analyze multiple samples simultaneously; expanded multiplex scales, delivering higher marker numbers per test; and improved sensitivity, reflected in more successful analysis of highly degraded DNA. Some of the current challenges encountered in forensic genetic casework are: 1) kinship analysis of increasingly complex pedigrees; 2) failure or reduced success when genotyping degraded DNA; and 3) the need to explore additional markers when no references samples are available to the investigators. Here we present results obtained from the use of MPS SNP panels in routine casework undertaken in our laboratory, which includes distant kinship analyses, testing of remains from missing persons and criminal casework. MPS analysis used an Ion S5™ Semicon- ductor Sequencer and results were routinely compared to previously obtained data from established CE tests. In all the above situations, the analysis of ID-SNPs panels improved the power of discrimination when routine STR analysis failed to provide sufficiently powerful weight-of- the evidence. SNP analysis also helped resolve cases where STRs failed due to low level DNA samples. Lastly, when no reference samples are available for comparison, SNPs are increasingly useful to produce valuable investigative leads such as predicted biogeographical origin and pigmentation phenotypes of the DNA donor. We report successful application of all these MPS- based SNP analyses.

512 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P431 - MASSIVELY PARALLEL SEQUENCING OF STRs HAS GAINED NUMBER OF TYPED MARKERS IN THE ANALYSIS OF DEGRADED DNA Su-Jin Bae1, Ye-Lim Kwon1,2, Mi Hyeon Moon1,2, Kyoung-Jin Shin1,2

1 Yonsei University College of Medicine, Department of Forensic Medicine, Seoul, Republic of Korea 2 Yonsei University, Brain Korea 21 PLUS Project for Medical Science, Seoul, Republic of Korea

Massively parallel sequencing (MPS) of STRs is promising in the analysis of degraded DNA because it could simultaneously amplify STRs with small sized amplicons. However, STR genotyping of degraded DNA using MPS method is still challenging and this kind of trial was rarely reported. In this study, we upgraded two in-house MPS panels that can analyze 23 autosomal STRs and 23 Y-STRs with amplicon size ranges of 77–253 base pairs by removing minor PCR interference and increasing PCR yield. A total of 20 DNAs were extracted from more than 50 years old skeletal remains and the mean concentration was 57 pg/ul and degradation index values ranged from 1.3 to 21.5. All samples were typed in duplicate for PowerPlex Fusion and Y23, and MPS method, respectively. The barcoded MPS libraries were prepared by two-step PCR method and sequenced on a MiSeq System. The length-based genotypes of each STRs were analyzed from FASTQ files using the STRait Razor v3.0. Genotype recover rate for 50 pg of 2800M DNA were > 95 % on MPS of both autosomal and Y-STRs. In the MPS data analysis, more than five markers on average were additionally typed than those of corresponding capillary electrophoresis (CE) analysis for the 20 degraded DNA. Moreover, increased success rates of STR typing by MPS method were observed in 15 autosomal markers with highest in Penta D followed by D22S1045, and 15 Y-STR loci with highest in DYS643. In conclusion, we could get gains in the number of typed makers by MPS of STRs for degraded DNA, which were mainly achieved in the long-length target in CE methods. Therefore, MPS of STRs with small sized amplicons is able to increase power of discrimination substantially in the identification of old skeletal remains.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 513 P432 - MAXIMIZE INFORMATION FROM YOUR MIXTURE SAMPLES USING A COMBINED AUTOSOMAL STR AND Y-STR MULTIPLEX SYSTEM Ann Macphetridge1, Kristy Lenz2, Dawn Rabbach2, Cynthia Sprecher2, Douglas Storts2

1 Promega Corporation, Genetic Identity - SBU, Madison, USA 2 Promega Corporation, Research and Development, Madison, USA

The VersaPlex™ 27PY System is a 6-color, 27-loci STR system for simultaneously amplifying and genotyping 23 autosomal loci, 3 Y-STR loci, and Amelogenin. It is intended for forensic casework and kinship STR analysis in regions that prefer the D6S1043 locus. In addition to D6S1043, the autosomal loci are CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, -30 D22S1045, Penta D, and Penta E. These loci offer Probability of Identity (PI) values of 4.4 x 10 for four combined American populations and 6.2 x 10-27 for the Asian American population [1]. This system will be the only D6S1043-containing multiplex that includes multiple Y-STR loci. The DYS391 locus is included for gender-verification of Amelogenin-null samples. DYS570 and DYS576 are included for their high gene diversity values [2]. These three Y-STR loci allow more confident determination of the number of male contributors in complex mixtures. While designed for an optimal input DNA amount of 1ng, it is sensitive to low DNA input amounts. The system is robust against high concentrations of PCR inhibitors. Nine loci are under 200bp, making it useful for degraded samples. The presentation will include data highlighting the kit’s capabilities. References [1] C. R. Steffen, M. D. Coble, K. B. Gettings and P. M. Vallone, “Corrigendum to ‘U.S. Population Data for 29 Autosomal STR Loci’ [Forensic Sci. Int. Genet. 7 (2013) e82–e83],” Forensic Science International: Genetics, pp. e36-e40, 2017. [2] J. Purps and L. Roewer, “A global analysis of Y-chromosomal haplotype diversity for 23 STR loci,” Forensic Science International: Genetics, pp. 12–23, 2014.

514 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P433 - MITOCHONDRIAL DNA DATA ANALYSIS STRATEGIES THAT INFORM MPS-BASED FORENSIC CASEWORK IMPLEMENTATION Cydne Holt1, Tim Hill1, Paulina Walichiewicz1, Kathryn Stephens1, Justin Eagles1, Anthony Rensfield1, Meghan Didier1

1 Verogen Inc., Forensic Science and R&D, San Diego, USA

Massively parallel sequencing (MPS) of human mitochondrial control region or whole genome is now faster, easier and more informative than was possible with Sanger sequencing and capillary electrophoresis. This presentation focuses on the aspects of mtDNA data that are important to consider when implementing MPS-based mtDNA typing into casework operations. The presentation will include evaluation data of the ForenSeq mtDNA typing system that indicate how interpretation concepts integral to Sanger sequencing of mtDNA apply directly to MPS-based mtDNA typing. In addition, there are new interpretation considerations that may be helpful when determining standard operating procedures and casework interpretation guidelines. Se- quencing data will be presented that may assist with SOP determination relative to the possibility of signal “cross talk” during sequencing and sample carryover between MPS runs. The presentation will also include a summary of approaches to sensitivity assessments that may help inform interpretation criteria, including thresholds, relative to input DNA template concentration ranges, number of pooled samples for MiSeq FGx sequencing runs and downstream bioinformatic demultiplexing. Given the quantitative nature of digital MPS data, these limit of detection studies may also assist when laboratories wish to set criteria for heteroplasmy detection (within an individual) or for mixed DNA sample interpretation. Assessment of variables will be provided across sample types such as hairs of different color, texture, environmental states, and PCR/sequencing inhibition when typing skeletal and dental remains to assist with fundamental understanding of ForenSeq mtDNA typing system limitations as well as metrics that may assist when making data quality determinations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 515 P434 - MPS REVEALS ISOMETRIC PCR ARTEFACTS IN DEGRADED SAMPLES Paolo Fattorini1, Barbara Gornjak Pogorelc2, Solange Sorçaburu-Ciglieri1, Jože Balažic2, Carlo Previderè3, Irena Zupanič Pajnič2

1 UCO of Legal Medicine, Department of Medicine- Surgery and Health, University of Trieste, Trieste, Italy 2 Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Section of Legal Medicine and Forensic Sciences, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy

It is well known that DNA damage promotes PCR artefacts. In addition, as MPS provides the fin- est typing of the PCR products, this technology should be able to evidence PCR artefacts that CE is not. To test this hypothesis, a DNA sample was degraded by heating at 70 °C up to 24 hours. The resulting samples #1, #2 and #3 showed a Degradation Index (as assessed by PowerQuant kit) of about 30, 180 and 500, respectively. One nanogram of each of the three degraded samples as well of the undegraded control was analysed by the Precision ID Globalfiler NGS STR Panel v2, in duplicate tests. The libraries were prepared by an Ion Chef™ System, loaded into an Ion 520 Chip and run on an Ion S5 sequencer. The data analysis was performed by Converge v2.1 using the default setting parameters and a minimum coverage of 100x for locus call. No artefact was found both in the control and in the less damaged sample #1. Well known artefacts, such as allelic imbalance phenomena, stutter products and allelic drop out phenomena were found with low frequency in samples #2 and #3. Drop in phenomena, flagged by the software as additional alleles, were scored with the remarkably frequency of 6.1–13.1 % in these two samples. The majority (18 out 22) of these PCR products were sequenced as “isometric artefacts” (IA). The molecular features of these IAs are the following: same length of the parental alleles; at least one nucleotidic substitution within the STR motives; coverage ratio with the parental allele of 0.1–0.9 (median value: 0.2). Thus, this study shows that IAs represent the most abundant class of PCR artefacts, which cannot be identified by the employment of CE. As IAs can mimic a mixture, the impact of their finding in real casework is discussed.

516 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P435 - NAKED EYE AMELOGENIN Y DETECTION USING DNAZYMES ON A µPAD TOWARDS RAPID GENDER DISCRIMINATION Enrique Azuaje-Hualde1, Susana Arroyo-Jiménez2, Gaizka Garai-Ibabe2, Marian M. De Pancorbo3, Fernando Benito-Lopez2, Lourdes Basabe-Desmonts4

1 University of the Basque Country, BIOMICs MICROFLUIDICS Research CLUSTER, Vitoria-Gasteiz, Spain 2 University of the Basque Country, Analytical Microsystems & Materials for Lab-on-a-Chip AMMa-LOAC Group- Microfluidics Cluster, Vitoria-Gasteiz, Spain 3 University of the Basque Country, BIOMICs Research Group, Vitoria-Gasteiz, Spain 4 University of the Basque Country, BIOMICs microfluidics Research Group, Microfluidics Cluster / Basque Foundation of Science- IKERBASQUE, Vitoria-Gasteiz / Bilbao, Spain

Gender determination can be particularly crucial in forensic caseworks. Differences in the amelogenin gene between Y and X chromosomes are often used for sex determination by PCR methods, but they require laboratory equipment and trained personnel (Tozzo et al., J Forensic Leg Med, 2013, 20(5): 387–391). Due to the variety of samples found at crime scenes, an in situ sex discrimination of the samples would help to classify them prior to the laboratory analysis [Ren et al., Curr Opin Biotechnol, 2014, 25: 78–85.). In this regards, microfluidic devices require small sample volume and can provide a rapid analytical result. With them, bare-eye colorimetric signals can be obtained (Saez et al., 15th IEEE Sen- sors Conference, 2017). This has created an interest on point-of-care (POC) devices, which deliver rapid results and can be handled by non-qualified personnel. Therefore, POC tests can reduce analysis cost and time (Abel G., Expert Rev Mol Diagn, 2015, 15(7): 853–855). The low cost production of paper devices has led to the development of POC analyses including colorimetric assays that can be suitable for sex discrimination. Catalytic abilities of DNA probes acting as DNAzyme allow nucleic acid detection when bind to specific genome sequences and hemin. The complex gains peroxidase activity, allowing the oxidation of ABTS salt in the presence of H2O2, producing green color (Garai-Ibabe Anal Chem, 2014, 86(20): 10059–10064). In this work, we propose a methodology for the detection of Y amelogenin gene on a microfluidics paper device using a DNAzyme that hybridizes specifically with Y amelogenin gene. The reaction would be carried out inside on filter paper, in order to create a paper device for the naked eye (green color apparition) determination.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 517 P436 - NEW APPROACH TO SIMULTANEOUSLY IDENTIFY MITOCHONDRIAL DNA HAPLOGROUPS BY MULTIPLEX PCR‑RFLPS AND CAPILLARY ELECTROPHORESIS German Burgos Figueroa1, Eliana Jazmín Chacon-Duran2, Marcelo López-Carrera3, Byron Freire- Paspuel2

1 Escuela de Medicina- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 Laboratorios de Investigación, Universidad de Las Américas UDLA, Quito, Ecuador 3 Laboratorio de ADN, Fiscalía General del Estado, Quito, Ecuador

Mitochondrial DNA (mtDNA) genotyping provides a valuable tool for forensic studies and population genetics. Nevertheless, current methodologies such as singleplex PCRs & RFLPs, SNaP- shot and Sanger Sequencing are time-consuming and expensive. Here we describe a detailed new multiplex PCR-RFLPs approach to simultaneously identify mtDNA haplogroups commonly present in Latin-American populations. The markers to determine haplogroups A, C, D, H and L were coding region SNPs that exhibit distinct restriction sites; meanwhile, a distinct 9bp-del mutation was chosen as a marker for the haplogroup B. These markers were selected from the Global mtDNA Phylogenetic Tree Build 17 (Feb 2016) proposed by van Oven & Kayser (2009). A set of FAM-labelled primers was designed for each haplogroup using Primer-Blast (NCBI) and verified for dimers and hairpins by Autodimer V1. Two multiplex PCR reactions were standardized. The first one for haplogroups A, C, D and H, and the second one for haplogroups B and L. The PCR products of the first PCR were double digested with the restriction enzymes HaeIII and AluI while the second PCR products were digested with HpaI. Both multiplex digestion products were mixed and separated by capillary electrophoresis in a single run to determine fragment sizes using Genemapper V3.2. The genotypes obtained by this multiplex PCR-RFLPs analysis were also confirmed by Sanger sequencing. Indeed, the results showed that this approach allows the simultaneous determination of mtDNA haplogroups commonly present in Latin-American populations in a relatively fast, accurate, and economical way. This offers a time-optimizing experimental framework as well as a better resolution and more sensitive detection procedure to assess mtDNA SNPs for population and forensic studies.

518 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P437 - NEW INSIGHT: WHOLE GENOME SEQUENCING DATA IN FORENSIC STR ANALYSIS Yifan Xie1, Fang Chen1, yanyan Zhang1, Yicong Wang1, guifang Yang1, Ning Wang1, Jianqiang Wu1

1 MGI-BGI-Shenzhen, Application research and development center, Shenzhen, China

Current short tandem repeats (STRs) were amplified by commercial and in-house multiplex PCR kits and analyzed by capillary electrophoresis (CE). Limited STRs as well as lacking sequence information made it hard to build a comprehensive population frequency database. In recent years, next generation sequencing (NGS) has demonstrated its discriminatory powerin forensic analysis of STR, for discovering both length and sequence polymorphisms. And whole genome sequencing (WGS) has been used in research and clinics. With the decreasing cost of sequencing and the improvement of national medical system, WGS for everyone is future trend. How to make better use of WGS to save costs and facilitate various fields, especially forensic science, is worth considering. However, the potential power of WGS in forensic fields still remain uncertain. Here we evaluated the performance of WGS in forensic STR analysis. About 30X WGS data of NA12878 on different platforms, including Hiseq2500, Novaseq6000, BGISEQ-500 and MGIS- EQ-2000, were recruited. And 106 forensic STR, including 68 autosomal STR and 38 X-STR, were analyzed. Allele, stutter and artefact were distinguished by a stutter model. After quality control, 32 and 20 STR were genotyped in single-end 100bp sequencing data on BGISEQ-500 and MGIS- EQ-2000, and 51, 52, 56, 70 STR were genotyped in single-end 150bp sequencing data on BGIS- EQ-500, NovaSeq6000, Hiseq2500, MGISEQ-2000. It’s interesting that 92 STR including 20 STR in CODIS were genotyped in single-end 400bp sequencing data on MGISEQ-2000. Lengths were CE-concordant and sequences were consistent on different platform. In conclusion, WGS may be a promising direction in the future of forensic fields. However, data privacy and ethics need to be considered.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 519 P438 - NEW SRATEGIES IN THE FIELD OF MIXTURE DECONVOLUTION Katja Anslinger1, Birgit Bayer1

1 Institute of Legal Medicine, Forensic Molecular Biology, Munich, Germany

The interpretation of mixtures often represents a real challenge for forensic scientists. With the methods currently used in routine, certain issues that couldn’t yet completely be solved repeatedly occur. The DEPArrayTM Technology (Menarini, Silicon Biosystems) is a new method, which was successfully applied for the deconvolution of cell mixtures via distinction on the basis of immunofluo- rescence. Pure cell pools as well as single cells can be separated from a mixture of different cell populations. In our studies, we combined the potential of this technology in terms of separating cells of a particular cell type as well as separating single cells from the belonging cell population with single cell STR-profiling in order to answer questions that could not yet be fully clarified. Thus, DNA profiles of each individual contributor could be reconstructed from mixtures com posed of equal amounts of cells belonging to the same type through the isolation and STR-profiling of multiple single cells. Moreover cases, in which the central question was whether a suspect really contributed blood rather then skin cells to a given mixture, could be solved. This technique also was used to clarify cases in which differential lysis led to inconclusive or non satisfactory results, as happened in cases of incomplete differential lysis or multiple rape. The technique itself, the validation but mainly impressive examples of routine case work samples (including unpublished data), where the superiority of this technique in comparison with the previously used methods is shown, will be introduced.

520 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P439 - NEXT GENERATION SEQUENCING TECHNOLOGY IN SECOND WORLD WAR VICTIM IDENTIFICATION Irena Zupanič Pajnič1, Barbara Gornjak Pogorelc1, Jože Balažic1

1 University of Ljubljana- Medical Faculty, Institute of Forensic Medicine, Ljubljana, Slovenia

DNA analyses of WWII skeletons have been used for identifying victims from mass graves found in Poland, Bosnia and Herzegovina, Croatia, Slovenia, Russia and others. The killings during the WWII, with nearly 100,000 victims, is one of the greatest losses of life in Slovenia’s modern history and most of the victims are still buried in hidden mass graves and remain unidentified. Identity, ancestry, and phenotypic SNPs, as well as STR markers are already used for solving various cases with NGS technology. In this study, the Precision ID GlobalFiler NGS STR panel was used to identify the WWII victim that could not be identified with CE analyses because limited statistical support was obtained after amplification of autosomal STRs using CE STR kits. Bones and teeth were analysed and compared to family references (nephew and niece on paternal line). Prior to DNA isolation 0.5 g of powder was decalcified. The DNA was purified in a Biorobot EZ1 device. The nuclear DNA of the samples was quantified with the PowerQuant kit. Because the recommended PP of 99.9 % was followed with the goal of high confidence of correct identification, the NGS STR Panel was used, and after the analysis of additional STR loci the statistical calculation showed a PP of 99.99986 %, showing that a large enough number of genetic markers were analysed when identifying the skeletal remains of the aunt. PP value endorsed the hypothesis that the tooth and bone samples were from individual related to the family references rather than from unrelated individual. In presented case NGS technology proved to be a powerful tool for increasing the number of autosomal STRs needed for identification of WWII victims when linear markers cannot be used for comparison and only distant relatives are available for analyses.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 521 P440 - NON-DESTRUCTIVE DNA COLLECTION FACILITATING A REVISED FORENSIC WORKFLOW FOR HANDWRITTEN DOCUMENTS Patrick McLaughlin1, Eliot Springer2, Mechthild Prinz1

1 John Jay College of Criminal Justice, Science, New York, USA 2 New York City Police Department, Crime Lab, New York, USA

Current forensic processes for handwritten documents aim at preserving probative indentations and latent fingerprints prior to DNA collection. However, developing indentations via ESDA will transfer DNA from the paper to the ESDA foil and fingerprint developers such as 1,2 indanedione may affect STR allele calling rates. We developed a dry vacuum trace DNA recovery technique that is non-destructive and can collect material deposited during the process of writing. A vacuum connection and a Carolina disposable 9-inch glass pipette blocked with a moistened (0.01 % TritonX) Puritan cotton swab were used to collect material from white printer paper. DNA was extracted with a modified Chelex method and processed using Applied Biosystems Quantifiler Trio and AmpflSTR Identifiler Plus. Ten volunteers donated fingerprints to be developed with magnetic black powder or 1,2 Indanedione. The vacuum process did not interfere with friction ridge detail. DNA recovery was evaluated using 11 volunteers who were asked to write a specified text on white printer paper. Another set of six volunteers provided the same writing sample before and after exercising. 17 % of the samples gave single source full profiles. Most of the samples (52 %) were mixtures, but of these 50 % had a CODIS eligible major component. For the vacuum method to be non-destructive the paper evidence must be held in suspension. For a full validation of this collection method other commonly encountered types of paper, such as notebooks, magazines, bank deposit slips and newsprint will be tested.

522 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P441 - NONINVASIVE PRENATAL PATERNITY TESTING (NIPAT) THROUGH MATERNAL PLASMA DNA SEQUENCING WITH A TWO-STEP MULTIPLEX PCR STRATEGY Guifang Yang1, Yifan Xie1, Lin Yang1, Yicong Wang1, Yu Zheng1, Dan An1, Yanyan Zhang1, Fang Chen1

1 MGI-BGI-Shenzhen, Application research and development center, Shenzhen, China

The frequently used technologies for noninvasive prenatal paternity testing (NIPAT) were short tandem repeats detection by capillary electrophoresis and SNP typing array with maternal plasma DNA. Previously, our lab has established a method through maternal plasma DNA sequencing by capturing over 5000 SNPs with probes. Although it has been validated effective and widely used in real setting, it’s low in capture rate and time consuming. In this study, we aimed to establish a simple and efficient method for plasma DNA-SNPs typing and validate its performance on NIPAT. We screened out 1206 SNPs with minor allele frequency above 0.35 and no linkage with each other based on the simulated model established by our lab. It is expected when the effective SNP loci (the SNP homozygous in mother) are more than 500 with a sequencing depth higher than 1000X, the fetal fraction limit of detection can be as low as 2 % for accurate paternity determination. All the amplicons were designed within 100 bp for efficient amplification of plasma DNA with a self-developed two-step multiplex PCR technology. Libraries of 8 pregnancies were sequenced on MGISEQ-2000 (SE50) to perform NIPAT. The results showed that the effective SNPs were more than 570 with a sequencing depth of 10000X for each pregnancy, and the detected fetal fraction was as low as 1.3 % at 6 weeks of gestation. All of the 8 pregnancies were correctly determined and the whole process was completed within 27h from sample to result. The results demonstrated that our simulated model is feasible, and the 1206X SNP panel can accurately perform NIPAT at fetal fraction above 2 %. In addition, our two-step multiplex PCR technology of amplifying thousands of SNPs could be a simple and efficient method for forensic applications in the future.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 523 P442 - OPTIMISING DNA RECOVERY Jarrad Rennie1, Adrian Linacre1, Paul Kirkbride1

1 Flinders University, College of Science and Engineering, Adelaide, Australia

We report on the use of a newly developed forensic swab that is specifically designed to both efficiently recover and release touch DNA samples. Currently swabs in use for the collection of trace touch DNA are effective at either removal of the DNA but retaining in subsequent processes, or poor at collecting the DNA from a substrate but effectively release the collected DNA at the next step. The aim of this project is to develop a swab that combines the best of both and collects DNA effectively then releases when needed. This study looked at foam, cotton, polyurethane and nylon-based swabs that are commercially available. In addition, we coated both the swabs and silica fibres with a chitosan polymer at an optimal concentration and pH and compared the recovery and release rate of each swab. The newly developed swab utilises a pH dependent chitosan coating and is antimicrobial to prevent bacterial growth. QIAamp DNA Investigator kit was used to show the recovery and extraction efficiency of each fibre from a known DNA concentration and from touch DNA samples. Results indicate that uncoated commercially available swabs were less efficient at recovering touch DNA. Those swabs coated in chitosan collected greater amounts of DNA than other swabs at varying degrees. The flocked nylon and silica fibre coated with the polymer were found to have best recovery and release efficiency of all swabs tested. The outcome shows that coated flocked nylon and silica fibres are promising materials worthy of further development towards a swab that both attracts DNA as a collector and then releases DNA into either a lysis buffer or PCR solution.

524 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P443 - OPTIMIZATION OF SINGLE CELL AMPLIFICATION AFTER DEPArray™ ISOLATION Valentina Meloni1, Laura Lombardi1, Roberta Aversa2, Andrea Berti1, Filippo Barni1

1 Carabinieri Corps, Reparto Carabinieri Investigazioni Scientifiche di Roma – Sezione di Biologia, Rome, Italy 2 Menarini Silicon Biosystems S.p.A., Research and development, Bologna, Italy

Single cell isolation in forensic genetics has been attempted for many years using different technological approaches such as FACS and LCM. More recently DEPArray™ digital sorting technology (Menarini Silicon Biosystems) was applied also in forensic genetics, for the isolation of pools of pure cells or single cells. This new technology enables the use of single cell analysis to resolve forensic mixed samples. The purpose of this work was to evaluate the performance of different kits and different PCR conditions for STR profiling of samples containing very few cells and down to the single cell level. To generate precise aliquots of cells, 100 ml of healthy donor blood were collected on a swab and prepared with DEPArray™ Forensic Sample Prep kit for cell sorting on DEPArray™ NxT; from there, replicate pools of precisely counted 10 WBC (n=20), 5 WBC (n=23) and multiple single WBC (n=20) were isolated. Recovered cells were then lysed using DEPArray™ LysePrep Kit, and amplified, by adding PCR reaction mix in the same tube of each lysed cell(s) recovery. We tested 3 commercial amplification systems: PowerPlex® ESX 17, PowerPlex® Fusion 6C (Promega Corporation) and AmpFℓSTR® NGM SElect™ (ThermoFisher Scientific), at different cycle numbers (from 29 to 32). Generated profiles were evaluated by several metrics: STRs profile completeness, peak height mean value, stutters percentage and artifacts, according to internal validated thresholds. Results show the possibility to obtain more than 65 % completeness from a single cell amplification. Data also show that there is not a single optimal amplification kit for different cell input: PowerPlex® ESX 17 seems to work better for single and 5 cells group, while PowerPlex® Fusion 6C performed better for 10 cells amplification.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 525 P444 - OPTIMIZATION OF THE COLLECTION AND ANALYSIS OF TOUCH DNA TRACES Hannah Tabea Haase1, Helle Smidt Mogensen1, Cathrine Bie Petersen1, Johan Frederik Petersen1, Anders Holmer1, Claus Børsting1, Vania Pereira1

1 Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Surfaces at crime scenes that were touched by the offender(s) or victim(s) are frequently swabbed in order to recover genetic material. These trace samples have often very low amounts of DNA and contain less DNA than the minimum requirement for PCR amplification used in our laboratory (200 pg). Nevertheless, touch DNA traces may significantly contribute to the outcome of a crime investigation and it is important to address if other genetic information may be retrieved. In this study, the sample collection efficiency of two types of nylon swabs (Copan) was compared to cotton swabs (Puritan) by collecting extracted DNA and touch DNA traces from different surfaces including gun shell casings. Different collection and extraction methods were tested (Proteinase K, Chelex, Sonication) as well as direct PCR. The challenging samples were amplified with the AmpFlSTR® NGM SElect™ kit and with the Precision ID mtDNA Whole Ge- nome Panel (Thermo Fisher Scientific). The results of the comparisons indicated that the regular type nylon swab (4N6FLOQSwab™) did not substantially increase the recovery of touch DNA compared to traditional cotton swabs. Higher yields, above 200pg, were obtained with the subungual type. In some cases, enough DNA was retrieved to generate full or partial STR profiles. Complete mtDNA profiles were obtained from fingerprint traces that did not result in full STR profiles with traditional methods.

526 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P445 - OPTIMIZED STRATEGIES FOR COLLECTION AND ANALYSIS OF MICRO TRACES Lisa Dierig1, Peter Wiegand1

1 Uniklinikum Ulm, Institute of Legal Medicine, Ulm, Germany

Collection and analysis of micro traces such as touch DNA present numerous challenges in forensic casework. Identification of potential depositing sites, poor visibility of skin cell conglomer- ates and low template samples are only some issues to be encountered. Selection of single skin flakes in order to avoid collection of DNA from multiple donors during so-called “blind-swabbing” is very time- and labor-intensive. Moreover, only few of those samples contain sufficient amounts of DNA for STR analysis. This research started with addressing the optimization of visibility and localization of those bioparticles. Microscopic examination of skin flakes on adhesive film was facilitated by optimal illumination using a stereo microscope with two opposite light sources. Staining of skin particles made illumination during microscopy even obsolete. Another crucial factor in micro trace analysis is DNA extraction. Many methods loose considerable amounts of DNA during purification. Comparison of different extraction methods showed that DNA purification can be neglected since PCR inhibitors are usually of no concern in micro traces. Therefore, direct lysis methods allow recovery of all available DNA in small extraction volumes. To approach the issue of mixture generation during blind-swabbing, it was further investigated, if narrowing down the swabbed area would help circumvent this problem but simultaneously increased the success rate compared to single skin flake selection. Taken together, optimization of those steps allows micro trace analysis to be less time- and labor-consuming and concurrently increases the success rate for STR typing.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 527 P446 - PATERNITY INCONSISTENCY AT CHROMOSOME 2 – AS AN EXAMPLE OF UNIPARENTAL DISOMY Andrzej Doniec1,2, Maria Wróbel1, Miłosz Januła1, Andrzej Ossowski3, Tomasz Kupiec1

1 Institute of Forensic Research, Forensic Genetic Section, Kraków, Poland 2 Institute of Zoology and Biomedical Research, Jagiellonian University, Department of Genetics and Evolotionism, Kraków, Poland 3 Pomeranian Medical University in Szczecin, Department of Forensic Genetics, Szczecin, Poland

DNA testing in cases of disputed paternity is a routine analysis carried out in genetic laboratories around the world. The purpose of the testing is to demonstrate similarities and differences in analyzed genetic markers between the alleged father, mother and child. The existence of differences in the examined loci between the child and the presumed father may indicate the exclusion of biological fatherhood. However, another reason for such differences is genetic mutations, including chromosome aberrations and genome mutations. The presented results relate to genetic analyses carried out on three persons for the purposes of disputed paternity testing; in these persons, a deviation from inheritance based on Mendel’s Law was found in 3 out of 47 STR-type systems examined. All polymorphic systems in which the paternity of the defendant was ruled out were located on chromosome 2. Additionally performed tests on 32 insertion-deletion markers (DIPplex, Qiagen) and sequencing of 94 polymorphic positions of the SNP type (Illumina, ForensSeq) did not exclude the defendant’s biological fatherhood. Analysis of STR allele sequences and their flanking regions confirmed the hypothesis that alleles on part of chromosome 2 of the child originated only from the mother. The results of the tests did not allow us to exclude the paternity of the defendant and are an example of uniparental maternal disomy, which is rarely described in the literature.

528 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P447 - PERFORMANCE AND CONCORDANCE OF THE FORENSEQ™ SYSTEM FOR THE ANALYSIS OF CHALLENGING SAMPLE TYPES Heather Mckiernan1, Yih Ling Saw1, Heather Milnthorp1, Phillip Danielson2

1 Center for Forensic Science Research & Education, Forensic Biology, Willow Grove, USA 2 University of Denver, Department of Biological Sciences, Denver, USA

The benefits of massively parallel sequencing (MPS) technologies for forensic applications has been well documented over the past several years. More recently, commercial assays and software, such as the ForenSeq DNA Signature Prep Kit (Verogen), have been specifically designed to address forensic needs. This study evaluated the performance of the ForenSeq workflow (ForenSeq™ DNA Signature Prep Kit, standard flow cell and MiSeq FGx™ Forensic Genomics Sys- tem) by examining multiple challenging sample types. A batch of 60 fired and unfired brass 9 mm rounds of ammunition were analyzed using in-house optimized protocols including soaking and sonication paired with organic extraction. Aliquots of sample extract (25mL each) underwent pre-amplification concentration and either analysis with CE (GlobalFiler™) or sequenced using the ForenSeq workflow (Primer Mix B). A direct comparison of Universal Analysis Software (UAS)-developed STR results to CE fragment length results showed 100 % genotype concordant results. Allelic dropout was noted with both systems, with similar rates of occurrence; however, an added 37 STR loci and 172 SNP markers were generated using MPS, enhancing the discriminatory power of results. Additionally, a panel of 30 challenging sample types encountered in forensic casework were created and analyzed in the same manner including laundered, aged, manually degraded, and touch samples (handguns and evidence from explosive devices), as well as low-level body fluid samples deposited on a variety of substrates with and without inhibitors. In addition to demonstrating full concordance with CE chemistries, these results will assist laboratories seeking to develop workflows for challenging sample types using the ForenSeq system.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 529 P448 - PERFORMANCE COMPARISON OF TWO MOST RECENT Y-STR MULTIPLEX SYSTEMS FOR REAL FORENSIC CASEWORK ANALYSIS Pankaj Shrivastava1

1 State Forensic SCience Laboratory- Sagar MP India, DNA Fingerprinting Unit, SAGAR, India

Detecting the presence of male DNA in a mixture is still a challenge in forensic DNA typing. The study presents a performance comparison of the two most recent Y-STR systems available for forensic casework namely Y filer plus (YFP) and PowerPlex Y 23 (PP Y23) multiplex system. The comparison was done using full and half reaction volumes of both the systems. On the basis of the work done on more than 200 case samples of sexual assault, it was observed that the PP Y23 multiplex system works better than YFP. In the experiments conducted with lesser reaction volumes, it was observed that even 8 μL PCR reaction mix of PP Y23 is able to detect 16 pg of male DNA; however, with YFP it was possible with100 pg. More balanced Y-STR profiles were observed with low male DNA with PPY23.

530 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P449 - PERFORMANCE EVALUATION OF THE PRECISION ID GLOBALFILER™ NGS STR PANEL V2 AND CONVERGE SOFTWARE V2.1 Theresa E. Gross1, Peter Schneider1

1 University of Cologne- Faculty of Medicine and University Hospital Cologne, Institute of Legal Medicine, Cologne, Germany

Massively parallel sequencing (MPS) technology provides an improved resolution of genetic variation in STRs with the possibility to detect sequence-based allelic variation in addition to length-based alleles. This will increase the number of possible alleles for specific STR loci and the power of discrimination, which can be of substantial value in complex kinship cases or mixture analysis in criminal casework. The Precision ID GlobalFiler™ NGS STR Panel v2 is a commercially available forensic multiplex of 31 autosomal STRs (20 extended CODIS, 9 additional MPS and two Penta STRs) and Amelogenin for the Ion S5™ System by Thermo Fisher Scientific. Several studies [1, 2] have assessed the performance of preliminary versions of this assay, but no evaluation of the final product has been published. We have evaluated the assay’s performance in regard to stutter and noise ratios, concordance (to CE data and between different S5 runs), sensitivity, inter- and intralocus balance as well as its applicability to casework and mixed DNA samples within the scope of the SeqForSTRs project (Sequencing of Forensic Short Tandem Repeats) – a collaborative effort of several police and university labs in Germany coordinated by the Federal Criminal Police Office (BKA). The results of our assessment are based on more than 200 DNA samples, which were manually prepared with an optimized MPS protocol for sequencing on the Ion S5™ System and subsequently analyzed with Converge Software 2.1 and in-house tools. This study was cofunded by the ISF-Programme of the European Commission. [1] Mueller et al., Forensic Sci Int Genet 36 (2018) 95–103. [2] Wang et al., J Forensic Sci 62 (2018) 1692–1703.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 531 P450 - PERUS WORKING GROUP: A DNA-LED MULTIDISCIPLINARY PROJECT FOR THE IDENTIFICATION OF POLITICAL MISSING PERSONS FROM THE 1970’S IN BRAZIL Samuel Ferreira1,2,3, René Huel4, Alexandre Raphael Deitos3,5, Marina Nogueira Di Giusto3, Ana Paula Tauhyl10, Marcos Paulo S. Machado3,6, Rimarcs Gomes Ferreira3,7, Isabela Maya W. Silva3, Talita Maximo C. Ribeiro3, Mariana Inglez3, Marina Gratão3, Maria Ana Correia3, Andersen Liryo3,8, Luciane Zanenga Scherer3,9, Maria de Fátima T. Guimarães3,10, Candela M. Barrio3, Ana Bilić11, Zlatan Bajunović12, Dijana Tanković12, Thomas Parsons12

1 Instituto de Pesquisa de DNA Forense- IPDNA-, Polícia Civil do Distrito Federal- PCDF, Brasília, Brazil 2 Comissão Especial sobre Mortos e Desaparecidos Políticos- CEMDP, Comissão Especial sobre Mortos e Desaparecidos Políticos- CEMDP, Brasília, Brazil 3 Grupo de Trabalho Perus- GTP, Grupo de Trabalho Perus- GTP, São Paulo, Brazil 4 International Commission on Missing Persons, ICMP- Koninginnegracht 12, The Hague, Netherlands 5 Surperintendência Regional, Polícia Federal em São Paulo, São Paulo, Brazil 6 Instituto Médico Legal, Polícia Civil do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 7 Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil 8 Museu Nacional, Universidade Federal do Rio de Janeiro- UFRJ, Rio de Janeiro, Brazil 9 Museu de Arqueologia e Etmologia, Universidade Federal de Santa Catarina- UFSC, Florianópolis, Brazil 10 Grupo de Trabalho Perus-GTP, Grupo de Trabalho Perus- GTP, São Paulo, Brazil 11 International Commission on Mission Persons, ICMP- Koninginnegracht 12, The Hague, Netherlands 12 International Commission on Missing Persons, ICMP- Koninginnegracht 12, The Hague, Netherlands

The Perus Working Group (PWG) was constituted in 2014 for identifying political missing persons who disappeared in the 1970’s during the military dictatorship in Brazil. PWG is a complex and challenging project involving highly degraded bone samples from more than 1,000 sets of skeletal remains exhumed from a clandestine mass grave found in the Perus cemetery in São Paulo in 1990. The PWG has a: a) management committee, composed by the Special Commission on Political Deaths and Disappearances, the Ministry of Human Rights, the Municipal Secretariat of Human Rights and Citizenship of São Paulo and the Federal University of São Paulo; b) scientific and identification committees, composed by forensic experts in anthropology, archaeology, genetics, legal medicine and odontology; and c) follow-up committee, composed by families of disappeared. PWG is conducted in partnership with the International Commission on Missing Persons (ICMP) that is responsible for the DNA analysis of bone, dental and blood reference samples, as well as DNA matching and consultation in case resolution. Anthropological analysis and DNA sampling in Brazil is used to prioritize cases that may correspond to any of the 41 high profile missing persons that may be present. ICMP’s success rate for obtaining STR profiles is over 90 %, on the hundreds of samples tested to date. Multidisciplinary evaluation is applied to each candidate DNA match, to jointly reach conclusions of identity, or to propose a path toward case resolution. Testing of multiple genetic systems, including ICMP’s large MPSplex SNP panel, is used in cases with deficient family references. These efforts, together with extensive effort in family outreach and archival case investigation has contributed to identifications early in the project.

532 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P451 - PRELIMINARY EXPLORATION OF A NOVEL METHOD FOR THE DECONVOLUTION OF DNA MIXTURES BY PYROSEQUENCING Yueyan Cao1, Ting Fang1, Yiwen Yang1, Qiuyue Wang1, Qiang Zhu1, Yuguo Huang1, Yuhan Hu1, Yijun Zhou1, Ji Zhang1*, Yufang Wang1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China

The deconvolution of DNA mixtures has been a thorny issue. Multiple genetic markers, such as STR, SNP, SNP/DIP-STR and microhaplotypes etc., as well as capillary electrophoresis, NGS and other technical platforms are used to solve this problem. We propose a novel method for the analysis of DNA mixtures. This method employed multiplex pyrosequencing to sequence four selected microhaplotypes.Via a sparse representation-based AdvISER-MH-PYRO algorithm, the original Pyrosequencing(PSQ) signals of DNA mixtures were deciphered to determine the contributors of the mixtures. Our initial exploration shows that this method can clearly resolve the DNA mixture composed of two individuals. The result shows that pyrosequencing signal analysis of DNA mixture can be used as an accurate, simple and low-cost method for the determination of contributors in DNA mixture.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 533 P452 - PREVALENCE AND IMPACT OF PCR ARTIFACTS FROM SOIL SAMPLES USING THE APPLIED BIOSYSTEMS™ GLOBALFILER™ PCR AMPLIFICATION KIT Toni Diegoli1, Tyiesha Moore1, Steven Weitz1, Todd Bille1

1 Bureau of Alcohol, Tobacco, Firearms and Explosives, Forensic Science Laboratory, Beltsville, USA

Forensic samples are rarely pristine, and may be collected from a variety of substrates containing unknown contaminants such as bacterial, fungal, or animal DNA. Unexpected artifacts are occasionally detected in commercial STR amplification kits despite incorporation of human-specific primers. Such artifacts are typically characterized during developmental validation and further through interrogation of customer reports so that they are known to the DNA analyst during interpretation of unknown profiles, as was the case for the Applied Biosystems™ Globalfiler™ PCR Amplification Kit. However, additional artifacts specific to samples contaminated with soil have been detected during routine use of this kit at the Bureau of Alcohol, Tobacco, Firearms and Ex- plosives Forensic Science Laboratory. A study was initiated to systematically characterize these artifacts and explore their prevalence in over 25 soil samples from locations across the United States. The impact of each artifact was assessed in relation to its location within the electropherogram (e.g. within a bin and/or within the allele-calling range) as well as its effect when amplified in combination with low-level human DNA. Approximately one third of the soil samples analyzed produced at least one detectable peak, some falling within allelic bins. Many of these artifacts are not listed within the published list of artifacts identified by the kit’s developers [1]. Possible strategies to mitigate the impact of such artifacts during interpretation of forensic casework will be discussed. [1] ThermoFisher Scientific Technical Note, Artifacts Identified Post-Developmental Validation: GlobalFiler™ PCR Amplification Kit, version February 12, 2019, available at: https://www. thermofisher.com/order/catalog/product/4476135.

534 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P453 - Q1a3a NATIVE-AMERICAN Y-HAPLOGROUP DETECTION IN DNA QUANTIFICATION STEP Santiago Ginart1, Mariela Caputo1, Daniel Corach1, Andrea Sala1

1 Universidad de Buenos Aires- Facultad de Farmacia y Bioquímica, Departamento de Microbiología- Inmunología- Biotecnología y Genética. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG - CONICET., Buenos Aires, Argentina

The knowledge of ancestral genetic information of a male contributor to a forensic sample can provide investigative clues such as assessing population of origin of the sample donor. Current- ly, this information is available after SNPs analysis. Some human DNA quantification kits might simultaneously provide information about gender or DNA degradation extent, but none supply information concerning genetic ancestry. Here we present a system of quantification of human DNA that detects Q1a3a native-American haplogroup (hg) by typing M3-Q3 C/T SNP. Detection is made by Real Time PCR followed by melting temperature (Tm) of one specific Y-chromosome fragment (58bp long) that determines Q1a3a hg (TmT=76 °C; TmC=77 °C=). We analyzed herein the usefulness of this diagnostic tool prior to STRs typing step. Fifty reference male DNA samples (blood, saliva and muscle from autopsy) were analyzed. DNA concentration ranged from

0,01 to 28 ng/uL. Thirteen were characterized as native-American (TmT = 76,02±0,04) while thirty-seven as non-native-American (TmC=77,13±0,06). These results were confirmed with Y-STRs haplotypes and White Athey´s hg predictor. Fifty casework samples containing male DNA were successfully haplogrouped and the STRs profiles, subsequently obtained, correlated well with quantitation results. The reaction cocktail also includes short (152 bp; Tm=84.9 °C) and long (244 bp; Tm=83.1 °C) fragments, allowing to infer DNA integrity. This system is a quick tool for choosing the most suitable Y-chromosome database for statistical evaluations and, simultaneously, allows quality evaluation of degraded samples. Since the detection chemistry is based on an intercalating fluorochrome, it is cost-effective and compatible with any real-time PCR platform that allows melting analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 535 P454 - QUANTITATIVE DETECTION OF SIGNATURE PEPTIDE IN BODY FLUIDS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS) Tebah Browne1, Marta Concheiro-Guisan1, Mechthild Prinz1

1 John Jay College of Criminal Justice, Science, New York, USA

Advances in mass spectrometry instrumentation have made universal peptide-based assays for body fluid identification more convenient and feasible. However, published peptide body fluid assays utilize specialized and expensive instrumentation that may be difficult to obtain for most crime labs. Adapting liquid chromatography tandem mass spectrometers (LC-MS/MS) for body fluid analysis maybe an alternative since many crime laboratories already utilize this instrumentation for toxicological analyses. In this study, we developed a quantitative method to detect multiple signature peptides in saliva and semen. Authentic liquid saliva and semen samples were digested using trypsin (Promega) in a 50 mM ammonium bicarbonate, 1 % ProteaseMax (Promega), 0.5 M dithiothreitol buffer. The trypsin digests were filtered and directly injected into the LC-MS/MS employing Thomson nano|Filter Vials®. Optimal conditions for the LC-MS/MS (Shimadzu LCMS-8050 Triple Quadrupole) were investigated employing peptide retention calibrators (Pierce) and synthetic peptides for semenogelin 1, submaxillary gland androgen regulated protein 3B, and histatin 1 (GenScript). Peptides were identified based on their retention time and the presence of two MRM (multiple reaction monitoring) transitions in electrospray positive mode. The target peptides were successfully detected in replicates from different donors (n=16) and specific for each body fluid. Peptides could be quantified by creating external calibration curves in a concentration range of 50–0.005 nmol/ mL. Sample preparation was fast and markers were combined in a multiplex. Peptide quantitation has several potential applications in forensic science, e.g. predicting downstream DNA typing success, or determining melanin content in hair.

536 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P455 - RECOMMENDATION OF GE.F.I. (ITALIAN SPEAKING GROUP OF ISFG) Loredana Buscemi1, on behalf of Gefi Group2

1 Polytechnic University of Marche, Azienda Ospedali Riuniti, Ancona, Italy 2 Gefi, Genetisti Forensi Italiani, Italy, Italy

Well-established workflows, based upon high level standards, are needed to assure reliability to the DNA evidence. To this aim, the board of the GeFI (Italian speaking group of the ISFG) developed recommendations on three topics of the Forensic Genetics in the last years: 1) personal identification tests. These recommendations were developed with the co-operation of both Polizia di Stato and Carabinieri and they were finally approved by the board of the ISFG; 2) paternity testing; 3) collection of the biological evidences in rape/maltreatment cases. These last recommendations were adopted by the Ministry of Health and published inside the “National guidelines for health agencies and hospitals on the subject of rescue and social-health assistance to women victim of violence” (Gazzetta Ufficiale n. 24 of 30, Gennaio 2018). The English version of the recommendation on personal identification will be available soon on the Ge.F.I. web site www.gefi-isfg.org.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 537 P456 - RESOLUTION OF mtDNA MIXTURES USING A PROBE CAPTURE NEXT GENERATION SEQUENCING SYSTEM AND CUSTOM ANALYSIS SOFTWARE Mary Wisner1,2, Shelly Shih2, Henry Erlich2, Cassandra Calloway1,2

1 Forensic Science Graduate Group, University of California Davis, Davs- CA, USA 2 Children’s Hospital Oakland Research Institute, University of California San Francisco Benioff Children’s Hospital, Oakland- CA, USA

One of the persisting challenges in the forensic DNA analysis field is interpreting DNA mixtures when the minor contributor falls below detection limits or when the DNA is degraded. Mixtures with roughly equal proportions (e.g. 50–50) also pose difficulties. Utilizing mitochondrial DNA (mtDNA) analysis can be beneficial due to the higher copy number per cell and the haploid nature of the genome. The conventional method for mtDNA analysis, Sanger sequencing, is insufficient for mixture analysis; haplotype assignment is nearly impossible due to overlapping data peaks and peak heights that do not always correlate to mixture proportions. The massively parallel and clonal sequencing aspects of Next Generation Sequencing (NGS) allow the analysis of the individual components of a mixture by counting sequence reads. We have used our custom mitochondrial genome probe capture NGS system to sequence complex mixtures. This target enrichment/NGS strategy can be valuable for detection of minor or multiple contributors and allow us to estimate the number and the proportions of the contributors. We report here analysis of contrived mixtures of two contributors in a 50:50 ratio, a 95:5 ratio, a three-person mixture, as well as a forensically relevant mixture. Finally, for data analysis, we use both variant frequency-based software GeneMarker®HTS and phylogenetic based software Mix- emt. GeneMarker®HTS allows us to detect private mutations whereas Mixemt software allows us to assign each sequence read to its contributor using phylogenetic information. Using our custom probe capture NGS system and both GeneMarker®HTS and Mixemt software, we can more reliably interpret complex mixtures that may be too degraded or limited for nuclear DNA analysis or mtDNA analysis by Sanger sequencing methods.

538 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P457 - SALTY OR SWEETY? ALTERNATIVES FOR BONE PRESERVATION ALONG EXTENDED PERIODS OF TIME Mariela Caputo1, Lucía Garrigos2, Santiago Ginart1, Daniel Corach1

1 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Génetica. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG - CONICET., Buenos Aires, Argentina 2 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Génetica. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG, Buenos Aires, Argentina

Sodium chloride is an effective human corpse tissue storage medium. Due to hygroscopic features and other phyco-chemical attributes of solid sucrose, we tested its efficiency as preservation medium. We used a set of 18 phalanges of a hand of female buried for over ten year in a sealed codin. After exhumation, 9 phalanges were preserved in sugar and 9 in salt, individually stored in 15 ml polypropylene tubes at room temperature for 4 years. DNA was extracted by three methods: a- total bone decalcification; b- demineralization and c- purification by a semi-automated process. Quantification was performed by fluorimetry (F), Plexor (P) and with an “in house” method (See poster 0670). Amplification was performed with Global Filer and detected in ABI 3500. DNA yield by method a- (F: 1.36±0.93ng/ul; P:0.23±0.18ng/ul) was higher than for b- (F:0,99±0.31ng/ul; P: 0,14±0.13ng/ul) or c- method (not detected). Amplification efficiency was determined by the percentage of complete markers (22 loci), the inter-loci balance _ILB-(Shan- non Entropy; SH: 3.09 as maximum) and intra-locus balance (mean local balance: MLB). The inter-loci balance was higher for the preservation in sugar than salt (SH: 1.69 vs 1.07) while MLB values did not show significant differences (0.88 vs. 0.89). Samples preserved in sugar showed 46 % of the complete markers, while for those kept in salt only 36 %. The complete amplification was 90 % for method a-, 26 % of method -b and 8 % for method c-. Degradation ratio correlates with the genetic profile quality. The method a- presented a higher ILB than method -b (SH 2.06, vs 1.42). Sugar, as an alternative conservative medium combined with DNA extraction by method a- showed an optimal strategy to obtain good quality genetic profiles.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 539 P458 - SeqforSTRs (SEQUENCING OF FORENSIC STRs) – PROJECT UPDATE Christian Sell1, Sabrina Achtruth2, Marc Trimborn2, Lutz Roewer3, Sascha Willuweit3, Walther Parson4, Theresa Gross5, Peter Schneider5, Petra Albrecht6, Michaela Gross6, Thorsten Hadrys7, Peter Wiegand8, Angelika Minawi1, Eva Schultheiss1, Jens Teodoridis1, Ingo Bastisch1

1 Bundeskriminalamt, DNA- Analytik, Wiesbaden, Germany 2 Landeskriminalamt Berlin, DNA-Analytik, Berlin, Germany 3 Charité-Universitätsmedizin Berlin, Forensische Genetik, Berlin, Germany 4 Medizinische Universität Innsbruck, Institut für Gerichtliche Medizin, Innsbruck, Austria 5 Universitätsklinikum Köln, Forensische Molekulargenetik, Köln, Germany 6 Landeskriminalamt Rheinland-Pfalz, DNA-Analytik, Mainz, Germany 7 Bayrisches Landeskriminalamt, Forensische DNA-Analytik, München, Germany 8 Universitätsklinikum Ulm, Forensische Genetik, Ulm, Germany

Massively Parallel Sequencing (MPS/NGS) holds great potential for forensic DNA analysis. Nu- merous publications indicate that MPS could lead to a significant increase of evidential value especially with mixed or degraded samples. As a consequence, the number of crime scene samples useable for investigative or court-use purposes could increase. Within the EU-funded project “SeqforSTRs (sequencing of forensic STRs)” we validate MPS and evaluate its added value. The project results aim to give advice for implementing MPS in the workflow of forensic laboratories. Successful application of MPS requires thorough validation of the technology to assess its accuracy, sensitivity and reproducibility. In addition, the concordance to established methods and between different MPS kits/platforms needs to be confirmed. Also, application of MPS data in reporting requires data for assigning a weight-of-evidence to sequenced loci. Therefore, we analyzed >800 reference samples, representing central European population. Furthermore, we cover the suitability of MPS to analyze difficult and standard samples in comparison to current methods. For data analysis we use manufacturer‘s software but also other tools allowing analysis of repeat and flanking regions. An essential part of our data analysis strategy is the discrimination between background or sequencing errors versus “real reads”. We present preliminary frequency data of STR allele sequences from our population study and data from artificial case work samples. Additionally, we show early results of sequencing error comparison study between the Ion S5 and the MiSeq FGx. These data were obtained by sequencing a single known sample multiple times on both platforms and counting deviations from the known allele as error.

540 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P459 - SERS-ACTIVE FORENSIC EVIDENCE SWABS FOR CONFIRMATORY IDENTIFICATION AND GENOTYPING OF HUMAN BODILY FLUIDS David Evanoff1, Brittania Bintz1

1 Western Carolina University, Chemistry and Physics, Cullowhee, USA

Fluid-specific serological tests employing immunological and biochemical indicators are typically used for screening potential forensic DNA evidence. Many methodologies lack sensitivity and specificity. Additionally, biological fluid identification can be expensive, laborious, and require sample consumption. Raman spectroscopy characterizes the inelastic light scattering indicative of molecular composition. Due to the low probability of Raman scattering, Raman analysis of small amounts of an analyte can be problematic. Surface-enhanced Raman scattering (SERS) is an extension of the Ra- man technique in which analyte signals can be enhanced by several orders of magnitude when on or near a nanostructured metallic surface. Several previous studies have shown that Raman spectroscopy is well suited for biological fluids because it is rapid, selective, and non-destructive. In the forensic lab, Raman analysis could lessen the number of serological tests performed on an evidentiary item since it can be used for simultaneous identification of all relevant biological fluids. However, when low laser excitation powers and/or fast analysis times are required, SERS may be more appropriate. In this presentation, a novel method to identify human bodily fluid on nylon-flocked swabs coated with silver nanoparticles is described. Swab fabrication, efficacy of SERS enhancement, and the spectral identification of semen, vaginal fluid, and mixtures are discussed. Likewise, DNA extraction and quantification from samples collected on the SERS-active swabs is described as well as a comparison of STR profiles obtained from SERS-active and plain swabs. This presentation will demonstrate that confirmatory identification using SERS is robust, sensitive, and does not affect downstream analyses.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 541 P460 - SIMPLIFIED, RAPID DNA EXTRACTION PROTOCOL FOR STR TYPING FROM BONES Cheng Ho Phua1, Laila Hasap2, Phuvadol Thanakiatkrai1, Thitika Kitpipit1

1 Prince of Songkla University, Applied Science, Hat Yai, Thailand 2 Prince of Songkla University, Molecular Biotechnology and Bioinformatics, Hat Yai, Thailand

Bones are one of the body parts most tolerant towards various environmental conditions, especially as unidentified remains found in several circumstances: mass disaster, terrorism, war, plague, etc. Typically, the most complex and time-consuming part of the STR typing process is DNA extraction, with many different methods developed, such as organic extraction, total demineralization, and silica-based column extraction. These protocols may take more than a day to complete. In such events where time is of the essence for human identification of the remains, a simple and fast but effective method is required. This study aimed to develop a simplified and rapid DNA extraction protocol from bone samples. Two bone types (femur and tibia, both fresh and degraded) were incubated in different chemicals (e.g. SLS, EDTA, proteinase K, DTT) with different functions to release DNA from the bone matrix. We also evaluated various incubation conditions and tested various “mid-extraction components”, such as the supernatant and pellet generated during the extraction steps. Different chemicals, incubation conditions, and “mid-extraction components” affected the purity and quantity of DNA extracted. Our final protocol only required two hours of incubation and 0.1g of bone powder. STR typing results gave high-partial to full profiles. The rapid protocol could be used when there is an urgent need of STR typing for human identification or when the sample amount is limited with a significant reduction in cost and time for investigation.

542 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P461 - SINGLE STEP STRATEGY FOR ESTIMATING UNKNOWN STR ALLELE FREQUENCIES IN A POPULATION Lucía Garrigos1, Mariela Caputo2, Daniel Corach2

1 Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología- Inmunología- Biotecnología y Genética. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG., Buenos Aires, Argentina 2 Universidad de Buenos Aires- Facultad de Farmacia y Bioquímica., Departamento de Microbiología- Inmunología- Biotecnología y Genética. Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas SHDG - CONICET., Buenos Aires, Argentina

Constant growth of forensic DNA databases require new STR markers to be developed, tested and validated. Aiming to simplify the process of obtaining allele frequency distributions of new STR loci included in new megaplexes, a single step protocol was developed. This goal was achieved by pooling samples of genetically unrelated donors (n =99), containing the same amount of DNA (total DNA =4.5ng/ul) as measured by fluorometry. Suitability of the method was tested by amplifying commercial kits: GlobalFiler (GF), Verifiler Express (VFE) and Y-filer plus (YFP). Amplicon detection were carried out in an ABI-3500 using POP-7 and a 50-cm capillary array. The allele peaks height were used to estimate allele frequency and compared with the actual frequency calculated by counting method of the individual pooled samples. The multinomial goodness of fit test was performed to testing statistical differences. In case of VFE, 4.5ng was the DNA amount that could best estimate the allele frequency distribution; 1ng for GF and YFP. For simple, complex and compound makers, the highest allele frequencies were correctly estimated (p >0.05) but in some cases, it was not able to detect alleles in very low frequencies 2/198 or bellow either with VFE or GF. In case of D12S391, either for VFE and GF, some microvariants with frequency of 2, 3 or 5/198 were not assigned although they were present in the profiles, probably due to POP7 resolution. In case of YFP, some alleles were not detected at DYS385. Even though, with some limitations, the proposed strategy probed to be fast and efficient for the major allele frequencies estimation. This simple approach generates an empirical distribution that allows estimating allele frequencies in a population by a single step assay.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 543 P462 - SlimDna – AN IN-HOUSE EXPERT SYSTEM FOR STR PROFILING Malin Sanga1, Linus Ek2, Samuel Boiso1

1 National Forensic Centre, Swedish Police Authority, Linköping, Sweden 2 Information Technology Department, Swedish Police Authority, Linköping, Sweden

At NFC, internally developed software for DNA analysis, SlimDna, is used. SlimDna is complementary to the LIMS and keeps track of samples, sample batches, instrument files and generated data. The system communicates with the LIMS, instruments and other software. SlimDna includes four modules (Analysis, Control, Administration and Frequency calculation) and covers the entire analysis process from DNA extraction to reporting approved STR DNA profiles to the LIMS. SlimDna is built with Microsoft Internet Information Services (IIS) as application server and the user interface consists of Windows clients. For data storage MySQL is used. The software has been implemented in different packages, STR typing the latest. The system has streamlined the STR typing by providing, among other functions, automated interpretation of STR profiles, databases for observed variant alleles as well as tracking and logging of negative and positive controls. STR EPG:s from analysis of crime scene samples (around 30 000 per year) are evaluated by SlimDna according to set rules concerning for example stutter ratio, heterozygote balance and number of alleles per marker in order to define the profile as a single source genotype or a mixture. The SlimDna output is compared to a manual evaluation before the results are reported in the LIMS. This process saves nearly one minute hands-on time per sample compared with the previous Excel-based solution. For reference samples, SlimDna evaluates the majority (55 %) of the samples without manual revision. SlimDna’s automated interpretation has proven to be very accurate and will soon replace more of the manual revisions. In the near future, we aim to develop SlimDna further, with for example mixture a module supporting the reporting officers in analysis.

544 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P463 - STUDY BY NEXT GENERATION SEQUENCING OF SUDDEN CARDIAC DEATH (SCD) Beatrice Boschi1, Irene Giotti1, Elisabetta Pelo1, Ugo Ricci2

1 Azienda Ospedaliero Universitaria Careggi, SOD Diagnostica Genetica, Florence, Italy 2 AOU Careggi SOD Diagnostica Genetica, Forensic Genetics Unit, Florence, Italy

Sudden cardiac death (SCD) is one of the most common causes of death; most SCD are related to secondary arrhythmias, to structural heart disease, or to primary electrical abnormalities of the heart. A significant number of SCD, especially among young people, are due to genetic heart disorders, both with structural and arrhythmogenic abnormalities. However SCD occurs also in patients with negative clinical history, autopsy is not always conclusive for a diagnosis. Recent technological advances in DNA sequencing have led to the commercialization of genetic testing now widely available in clinical practice. In particular, next generation sequencing allows the large-scale and rapid assessment of entire genomes. Analysis of SCD with a NGS panel of 174 genes was performed in our laboratory in order to identify the genetic causes and thus to direct the clinician to an accurate clinical and genetic screening of relatives. Here, we show two SDC cases in which the application of the NGS protocol resolved the diagnosis. Case 1: male, 57, without story of syncope and no previously highlighted cardiac alteration, died post cardiac arrest; negative family history. Autopsy was apparently negative. Case 2: male, 52, who died during a football game; negative family history, neurological episodes occurred before death was reported by close relative. Autopsy was positive for ventricular hypertrophic.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 545 P464 - THE APPLIED BIOSYSTEMS™ GLOBALFILER™ IQC PCR AMPLIFICATION KIT: A 24-PLEX STR CASEWORK KIT WITH AN INTERNAL QUALITY CONTROL SYSTEM ENHANCEMENT Matthew Ludeman1, Robert Green1, Vivian Nguyen1, Robert Eardley1, Marc Short1, Julio Mulero1

1 Thermo Fisher Scientific, Human Identification, South San Francisco, USA

The GlobalFiler™ kit, first introduced in 2012 and now widely used in forensic DNA genotyping analysis, is a six-dye 24-plex optimized for the analysis of forensic casework samples that includes 21 autosomal STR loci plus three Y-chromosome markers (Y-indel, Amelogenin and DYS391). A useful new feature that has been included in other more recently-developed Applied Biosystems STR kits, such as the NGM Detect™ and VeriFiler™ Plus kits, is an Internal Quality Con- trol system (IQC). The IQC system consists of two synthetic markers (IQC Small and IQC Large), which are amplified with FAM dye-labeled PCR primers from a synthetic DNA template added to the Primer Mix kit component. These IQC markers are designed to bracket the read region at the small and large molecular weight ends (approximately 70 bp and 450 bp) of the electropherogram, and provide a positive signal that confirms proper amplification and electrophoresis in non-compromised human DNA-positive or human DNA-negative samples. Additionally, the IQC system is a sensitive indicator for the presence of PCR inhibitors that allows for the peak- height ratio of the IQC Large to the IQC Small marker to be used to distinguish between normal reactions, reactions with residual PCR inhibitors and those with degraded DNA. To leverage this new form of enhanced functionality, we have modified the original GlobalFiler kit to include the IQC system. We report here results from our development and validation of the resultant new GlobalFiler IQC kit. This kit will complement the existing GlobalFiler kit, providing the same multiplex STR functionality and power of identity with the added enhancement of an IQC system. For Research Forensic or Paternity Use Only. Not for use in diagnostic procedures.

546 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P465 - THE BEST POSSIBLE RESULT FROM THE MINIMUM AVAILABLE Ugo Ricci1, Anna Lucia Nutini2, Francesca Gerundino3, Beatrice Boschi3, Elisabetta Pelo3

In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory. To achieve this goal the quantification of extracts before typing plays a fundamental role. Es- pecially when touched objects need to be examined tens or hundreds of nanograms may be present, but also no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination. Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Finally, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also shown.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 547 P466 - THE DEVELOPMENT AND EVALUATION OF AN NGS PANEL INCLUDING 42 AUTOSOMAL STRS Qingxia Liu1, Qingqing Du1, Guanju Ma1, Chaolong Lu1, Lihong Fu1, Guangping Fu1, Qian Wang1, Bin Cong1, Shujin Li1

1 Hebei Key Laboratory of Forensic Medicine, Forensic Medicine, Shijiazhuang, China

Using capillary electrophoresis (CE), more than 20 short tandem repeats (STRs) can be co-amplified in a single reaction. With higher-throughput, next generation sequencing (NGS) supply a feasible technique to detect much greater markers simultaneously. In this study, we develop an NGS panel including 42 commonly used autosomal STRs (D1S1656, CSF1PO, D10S1248, D10S1435, D11S2368, D12S391, D13S317, D13S325, D14S608, D15S659, D16S539, D17S1290, D18S51, D18S535, D19S253, D19S433, D20S470, D21S11, D21S1270, D22-GATA198B05, D2S1338, D2S441, D3S1358, D3S1744, D3S3045, D4S2366, D5S2500, D5S818, D6S1043, D6S477, D7S1517, D7S3048, D7S820, D8S1132, D8S1179, D9S925, FGA, PentaD, PentaE, TH01, TPOX, vWA) and amelogenin on Illumina MiSeq FGxTM. Sequencing accuracy is validated by the sequence consistency of 2800 Control DNA detected using Sanger sequencing. The nomenclature incompatibility is found between the NGS-STR and CE-STR for 9 STRs (D3S3045, D6S477, D7S3048, D9S925, D14S608, D17S1290, D18S535, D21S1270, GATA198B05), despite of the correct sequences. To be consistent with the nomenclature of CE, we revise the motif repeat numbers of these 9 STRs according to sequence alignment. The panel performs good repeatability. Using the panel, we detected 59 unrelated individuals of Chinese Han population lived in Hebei province. The results of 2535 loci of all 2537 loci in 59 samples (99.92 %) are consistent with CE-STR typing.

548 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P467 - THE EFFECT OF SURFACE TYPE, COLLECTION AND EXTRACTION METHODS ON TOUCH DNA Salem Alketbi1, William Goodwin2

1 Dubai Police and University of Central Lancashire, Assistant Expert at General Department of Forensic Science and Criminology in Dubai Police, Dubai, United Arab Emirates 2 University of central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom

Over the last few years, trace DNA profiling technology was a challenging endeavour, nevertheless, with advancement in biotechnology, the sensitivity of DNA profiling has increased to a point of generating usable results for DNA interpretation. This has proved useful in investigation of many serious crimes, such as terror attacks, homicides, and burglaries. However, studies on the recovery and extraction efficiencies in the interpretation of touch DNA are still under researched. The following paper delves into investigating how different DNA collection (cotton swab, nylon flocked swab and SceneSafe Fast™ minitape) and extraction methods (PrepFil- er Express BTA™ and QIAamp® DNA Investigator) affects DNA profiling, as well as the recovery of touch DNA on a range 0f porous and non-porous surfaces when left on surfaces at room temperature. The initial results from this study show that the amount of DNA collected from the different surfaces is significantly affected by the type of surface (F5,36 = 3.469, p < 0.05) and the type of extraction method used (F1,36 = 72.286, p < 0.05). Keywords: Forensic science, Trace DNA, Touch DNA, DNA recovery, Nylon swab, Flocked swab, Cotton swab, Scenesafe FAST minitapes, DNA extraction, QIAamp DNA investigator kit, PrepFiler Express BTA, AutoMate Express, Quantifiler™ Human DNA Quantification Kit.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 549 P468 - THE EFFECT OF TIME AND ENVIRONMENTAL CONDITIONS ON TOUCH DNA Salem Alketbi1, William Goodwin2

1 Dubai Police and University of Central Lancashire, Assistant Expert at General Department of Forensic Science and Criminology in Dubai Police, Dubai, United Arab Emirates 2 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom

Trace or Touch DNA analysis has become an important aspect of a forensic laboratory’s workload and a crucial tool for investigators in many cases. However, there is a lack of research regarding the influence of environmental conditions on Touch DNA, which has proven to reduce traces of biological material in samples. Therefore, this study investigated the influence of time between deposition and recovery of Touch DNA, as well as the impact of temperature and humidity on a range of porous and non-porous surfaces. The initial results indicate that the amount of collected DNA from the selected surfaces was significantly affected by surface type (F3,24 = 55.91, p < 0.05), surface conditions (F2,24 = 12.34, p < 0.05) and time between deposition and recovery (F1,24 = 14.50, p < 0.05). Keywords: Forensic science, Trace DNA, Touch DNA, DNA recovery, Nylon swab, Flocked swab, Cotton swab, Scenesafe FAST minitapes, DNA extraction, QIAamp DNA investigator kit, PrepFiler Express BTA, AutoMate Express, Quantifiler™ Human DNA Quantification Kit.

550 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P469 - THE IMPACT OF FDSTools NOISE CORRECTION ON THE ANALYSIS OF DATA FROM THE ForenSeq DNA SIGNATURE PREP KIT Kristiaan van der Gaag1, Jerry Hoogenboom1, Corina Benschop1, Anouk Backx1, Sofia Zuniga1, Loes Busscher1, Rick de Leeuw2, Peter de Knijff2, Titia Sijen1

1 Netherlands Forensic Institute, Division of Biological Traces, The Hague, Netherlands 2 Leiden University Medical Center, Human Genetics, Leiden, Netherlands

Massively Parallel Sequencing (MPS) enables the simultaneous analysis of a much larger number of forensic markers compared to conventional capillary electrophoresis (CE) sizing-based genotyping. Sequencing not only reveals the length variation of short tandem repeats (STRs), but also sequence variation that can reside within the STR region or the flanks. This additional variation increases the discriminatory power of the STR markers substantially and can help to distinguish genuine alleles from stutter artefacts. However, even with MPS, STR stutters can impact the detection and retrieval of a minor contributor in mixed samples. MPS data of 350 reference samples analysed through the Forenseq™ DNA Signature prep kit (Verogen) were also analysed with FDSTools to categorise systemic noise, which can result from the amplification or sequencing process. Systemic noise includes STR stutters and recurring products from polymerase slipping at mono nucleotide stretches. After categorising this systemic noise for individual alleles in reference samples, FDSTools can apply noise correction to trace samples by filtering marked noise reads and adding them to the parent allele reads. Noise correction has two goals: 1) explicate the actual sequence variation in a sample and 2) improve the read count representation of the actual mixture composition. Noise correction was applied for all 60 STR and 166 SNP fragments residing in the ForenSeq™ DNA Signature Prep Kit (DNA primer set B) on a large set of purposeful DNA mixtures consisting of up to five contributors. We will discuss the impact of noise correction on analysis thresholds and the efficacy of retriev- ing alleles of a minor contributor in a mixture.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 551 P470 - THE MODIFICATION OF CHELEX-100 DNA EXTRACTION METHOD Duo Peng1, Zhilong Li1, Huan Tian1, Yuqing Liu1, Lu Yin1, Peng Bai1, Weibo Liang1, Lin Zhang1, Hui Wang2

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Chengdu Public Security Bureau, Department of Forensic Genetics, Institute of Forensic Science, Chengdu, China

The DNA extraction from biological samples is an important procedure in forensic science since the following STRs profiles provide information to personal identification. Although there are many commercial DNA extraction kits, chelex-100 extraction method is still commonly used in forensic practice due to its simple procedures and fairly low cost. The routine chelex-100 method includes sample processing, incubation, boiling and centrifugation. The whole process of chelex-100 DNA extraction method costs about 1.0 h – 1.5 h. Considering the tolerance of PCR inhibitors with commercial forensic STR kits, we modified the chelex-100 DNA extraction method by soaking samples with RNase-free water and the incubation was omitted. We then evaluated the optimized and conventional chelex-100 DNA extraction method with STR loci (AGCU Expressmarker22 kit) on an Applied Biosystems 3130 Genetic Analyser. The data were then analyzed by GeneMapper ID software. The results showed that the samples were correctly genotyped with our modified chelex-100 DNA extraction method. It is important to note that the modified method without protease K digestion cost only about 20 min. Interestingly, the supernatant fluid after centrifugation also contained mRNA, such as house-keeping gene GAPDH and blood specific mRNA marker HBB. All these results demonstrated that the modified chelex-100 DNA extraction method was simpler and more efficient, which would save time, manpower and financial resources in forensic practice.

552 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P471 - THE NEED FOR AUTOMATION IS LIMITED WHEN USING A QUICK AND INEXPENSIVE ONE-TUBE DNA EXTRACTION PROTOCOL FOR CRIME SCENE SAMPLES Christina Forsberg1, Linda Jansson1,2, Carina Ansell1, Johannes Hedman1,2

1 National Forensic Centre, Swedish Police Authority, Linköping, Sweden 2 Applied Microbiology, Department of Chemistry, Lund University, Lund, Sweden

Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticat- ed automation solutions. We have developed, validated and implemented a quick, inexpensive and efficient one-tube direct lysis DNA extraction protocol for the majority of our crime scene samples. The method includes one pipetting step, the addition of a lysis buffer based on Chelex beads, Proteinase K and Tween 20. After three incubation steps with vortexing in between, the sample is ready for downstream applications (DNA quantification and STR profiling). The amount of lysis buffer added varies between 200 – 1000 µL depending on the amount of carrier material in the tube. If needed the extract can be purified or concentrated using a filter device, in our case Amicon Ultra-2. Through in-house validation we show that the method is fit-for- purpose for application in casework for samples containing blood, saliva, shed cells and semen as it provides high DNA yields and amplifiability. The method was implemented at the Swedish National Forensic Centre (NFC) in February 2018. During the first year more than 35,000 crime scene samples were extracted using the method, generating DNA yields equivalent to the previously used method. In conclusion, the need for automation of the DNA extraction process is limited when using a one-tube direct lysis protocol including only the addition of lysis buffer and no transferring steps. In addition it saves costs as there is no need for expensive pipetting robots or service fees and since the reagents used in this direct lysis protocol are inexpensive.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 553 P472 - THE SUCCESSFUL USE OF CARTILAGE AND MUSCLE, AFTER 23 DAYS OF INHUMATION, FOR DNA TYPING. AND IDENTIFICATION OF TWO STILLBORN EXCHANGED AT A HOSPITAL Samuel Ferreira1, Arthur E. Svidizinski1, Marinã R. do Amaral1, Maria Emilia E. Siqueira1, Márcia Schelb1, Maitê C.M. Kessler1, Loyane C. D.Medeiros1, Rubiane Y.A. Lobo1, José Gerardo P. Pierre Filho1, Adriana V. Moraes1

1 Instituto de Pesquisa de DNA Forense- IPDNA, Polícia Civil do Distrito Federal- PCDF, Brasília, Brazil

In DNA testing for identification of deceased bodies, the kind and quality of source of samples along with the preservation of samples play an important role in the results of DNA typing and, thus, in the identification. In this work, we will present a successful case of using cartilage and muscle, after 23 days of inhumation, for DNA typing and the identification of two stillborn exchanged at a hospital, due to administrative mistakes, in Brasilia, Brazil. One stillborn had been buried in a cemetery. He was exhumed 23 days after his inhumation and was at advanced stage of the decomposition. The other stillborn was at the mortuary of the hospital in a refrigerator. In both bodies, we collected cartilage and muscle samples from knee joint and started DNA extraction at the same day. We used EZ1 DNA Investigator Kit for DNA extraction. DNA was purified in a Biorobot EZ1 (Qiagen). We used Plexor kit (Promega) for quantification and Globalfiler kit (Applied Biosystems) for amplification. DNA analysis was performed on an ABI 3500 Genetic Analyzer (Applied Biosystems). In both bodies, full profiles were obtained from all cartilage and muscle samples. Cartilage samples had higher DNA yields than muscle samples as we supposed to be according to our previous experience with these kind of samples and due to the fact that cartilage is an avascular tissue and, thus, resists the decomposition of body and DNA degradation for longer periods of time, comparing to muscle. We used buccal swabs as reference samples from the two couples to confirm the paternity and maternity in both cases. In this work, we would like to highlight the importance of soft tissues, especially cartilage, as an excellent source of sample for DNA typing in human identification in cases of bodies in decomposition.

554 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P473 - THE VALIDATION OF DNA/RNA COEXTRACTION USING STR LOCI AND mRNA MARKERS WITH FORENSIC PURPOSE Duo Peng1, Zhilong Li1, Huan Tian1, Yuqing Liu1, Lu Yin1, Hui Wang2, Weibo Liang1, Lin Zhang1, Peng Bai1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Institute of Forensic Science, Chengdu Public Security Bureau, Department of Forensic Genetics, Chengdu, China

Both personal identification and body fluids identification play important roles in forensic science field. Currently, DNA markers such as STRs and SNPs are widely used in personal identification; RNA markers such as mRNA and microRNA are commonly used to identify the nature of samples. However, DNA and RNA are separately extracted from biological samples, which is time-consuming and inefficient especially when there are too many samples to be detected in forensic practice. The Bioteke DNA/RNA coextraction kit makes it possible to extract both DNA and RNA in one tube simultaneously, so here we attempt to validate extracted DNA and RNA part from the blood with forensic purpose. The STR loci of AGCU Expressmarker22 kit were used to validate the DNA part and blood specific mRNA markers (HBB, GlycoA and ANK1) and house-keeping genes (GAPDH) were used to validate the RNA constituent. Besides, the accuracy of STR profiles was compared with that of chelex-100 DNA extraction method. After detecting STR loci and mRNA makers, GeneMapper ID software was used to analyse the data obtained from Applied Biosystems 3130 Genetic Analyser. The results showed that full and correct STR profiles were obtained from DNA/RNA coextraction kit. For RNA part, blood specific markers and reference genes were detected using the coextraction kit. Therefore, we preliminarily proved that the blood extract by Bioteke DNA/RNA coextraction kit was suitable for forensic personal identification with STR loci and body fluids identification with mRNA markers.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 555 P474 - THE YARA GAMBIRASIO CASE: COLLECTION STRATEGY AND DNA MASS SCREENING USED TO FIND THE PERPETRATOR DNA IN A CHALLENGING SCENARIO Nicola Staiti1, Fabiano Gentile1, Elena Pilli2, Giampietro Lago3

1 Reparto Carabinieri Investigazioni Scientifiche di Parma, Sezione Biologia, Parma, Italy 2 Reparto Carabinieri Investigazioni Scientifiche di Roma, Sezione Biologia, Roma, Italy 3 Reparto Carabinieri investigazioni Scientifiche di Parma, Reparto Carabinieri investigazioni Scientifiche di Parma, Parma, Italy

In 2011 a corpse of a teenager was found in a fallow field after three months of environmental exposure. The autopsy showed no sign of rape but the presence of several cuts on the victim’s body. Despite exposure to weathering and animals, the clothes from the waist up were well preserved, whereas the trousers were extensively torn and the underpants were clearly cut. Searching evidence from the perpetrator was challenging since the traces present on the victim’s clothes and underpants were subjected to morphological and chromatic modifications caused by the environmental exposure. At first, sample collection was based on an integrated approach that involved circumstantial information and observation with the forensic light. The use of this light source allowed us to identify some areas with more clearly fluorescence, also visible to the naked eye, that were the first collected. The inspection led us to perform approximately 300 samplings on the clothes. In one of these areas, the results of the first sampling showed the presence of alleles unrelated to the victim. As a result, an extensive sampling through the application of a virtual grid was undertaken in this area to confirm and improve the male profile and obtain serological information. Due to the complete absence of other relevant clues on the perpetrator, when a full male profile -Unknown Male #1 (UM#1)- was found on the victim’s clothes, a huge mass screening that involved the collection and analysis of over 16,000 reference samples was undertaken. After several months of investigation, the biological father of UM#1 was found. Since legitimate sons doesn’t match and the man died in 1999, the mass screening continued on searching the biological mother and, finally, the full match with the perpetrator.

556 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P475 - TILED AMPLICON mtDNA SEQUENCING USING PID NGS SYSTEM AND CONVERGE™ ANALYSIS SOFTWARE: A ROBUST AND SENSITIVE ASSAY FOR FORENSIC CASEWORK APPLICATIONS Jessica Lim1, Sharon Wootton1, Chantal Roth1, Ryo Hasegawa1, Chien-Wei Chang1, Sharada Vijaychander1, Jie Deng1, Matt Gabriel1, Angela Lackey1, Robert Lagacé1

1 Thermo Fisher Scientific, Human Identification Division HID - MPS, South San Francisco, USA

In forensic casework, mitochondrial DNA (mtDNA) is useful in the context of recalcitrant samples that fail to produce a standard STR profile. Traditional Sanger sequencing using capillary electrophoresis (CE) compels a limitation of sequencing of the mtDNA genome to the hypervariable region as sequencing of the whole mitochondrial genome (mtGenome) is both time consuming and cost-prohibitive. With the availability of massively parallel systems (MPS), the mtGenome can easily be prepared and sequenced using a tiled amplicon multiplex of 162 amplicons. Addi- tionally, the forensic mtDNA analysis module developed on ConvergeTMSoftware and optimized specifically for the Precision ID Control Region and Whole Genome panels provides streamlined analysis for haplotype and haplogroup designations as well as robust detection of nuclear mitochondrial DNA segments (NUMTs) and point and length heteroplasmies. DNA from samples with known haplotypes were obtained through Coriell and NIST. Libraries were prepared on the Ion Chef using the Precision ID mtDNA Whole Genome Panel and sequenced on the Ion S5. Reads generated on the system were aligned and compared to the rCRS and were evaluated for concordance, amplicon coverage uniformity, and presence of artifacts, heteroplasmies, and NUMTs using the mtDNA analysis module on Converge.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 557 P476 - TISSUE STORAGE SOLUTION FOR PRESERVATION AND TRANSFER OF FORENSIC SPECIMEN IN HIGH AMBIENT‑TEMPERATURE Thant Zin1, Achirapa Bandhaya1, Nathinee Panvisavas2

1 Faculty of Science- Mahidol University, Forensic Science Unit, Bangkok, Thailand 2 Faculty of Science- Mahidol University, Forensic Science Unit / Department of Plant Science, Bangkok, Thailand

In hot climates, quality of forensic tissue sample can be easily affected by the high ambient- tem- perature, i.e. between 30–45 °C, during evidence transfer. Temperature of an item placed under direct sunlight can increase up to 10–15 °C from the actual ambient temperature. In this study, 3 tissue storage solutions were compared; (1) DMSO buffer, (2) Longmire’s buffer, and (3) trehalose solution. Pieces of 300-mg porcine tissue were preserved and kept at 25, 40, 60, and 80 °C for 1, 2, 3, and 4 weeks. DNA analysis by PCR was conducted by using the β-actin nuclear DNA marker set (148, 211, 289, and 366 bp), and the mitochondrial cyt B DNA marker set (161 and 323 bp). Results showed that DNA in tissue was best preserved in DMSO buffer. Although samples preserved in Longmire’s buffer solution gave DNA analysis results for temperatures up to 60 °C, amplification results between replications were not reproducible. For those tissue samples preserved in trehalose solution, DNA markers larger than 300 bp were absent and irreproduc- ibility of amplification results were detected at a higher level when the storage temperature increased from 25 °C to 40 °C, 60 °C, and 80 °C, and storage period was prolonged over 2 weeks. Results suggested that storage temperature at 80 °C and prolonging of sample storage over 1 week is not recommended. Experimental results here provided an alternative collection and preservation method for tissue samples at ambient temperature (without cold-storage) for subsequent DNA analysis, which can potentially be implemented in forensic biological evidence collection, preservation and transfer in hot-climate countries.

558 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P477 - TOWARDS INCREASING THE EFFECTIVENESS OF RECOVERING, IDENTIFYING AND SEPARATING SPERMATOZOA PRESENT ON VAGINAL SWABS TAKEN IN SEXUAL ASSAULT CASES Tim Clayton1, Finlay Kennedy2, Dan Beaumont3, Jim Thomson4

1 Eurofins Forensic Services, Casework Exam and Reporting, Wakefield, United Kingdom 2 Eurofins Forensic Services, Casework Exam and Reporting, Risley, United Kingdom 3 Eurofins Forensic Services, Casework Exam and Reporting, Culham, United Kingdom 4 Eurofins Forensic Services, DNA Research and Development, Teddington, United Kingdom

In 2017, our laboratory began an 18 month continuous improvement study designed to increase success rates in sexual assault cases targeting, in particular, vaginal swabs where the amounts of sperm present were relatively low. The objectives of the project were threefold (1) to optimise the recovery of semen from vaginal swabs, (2) to reduce the time spent searching microscope slides to identify sperm and (3) to reduce the incidence of contaminating female DNA in the seminal fraction following a differential extraction. We report the outcomes of the project and the consequent implementation of a modified sex swab protocol into our laboratories. This modified protocol which we have called ‘SpermTrap’ has improved success rates with intimate swabs containing low numbers of spermatozoa.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 559 P478 - USING REAL TIME PCR AS STRATEGY TO EVALUATE PERFORMANCE OF PCR AND SANGER SEQUENCING REACTIONS German Burgos Figueroa1, Byron Freire-Paspuel2

1 Escuela de Medicina- Facultad de Ciencias de la Salud, Universidad de Las Américas UDLA, Quito, Ecuador 2 Laboratorios de Investigación, Universidad de Las Américas UDLA, Quito, Ecuador

Sanger sequencing is an indispensable technique in forensic science. However, it is a time consuming process that requires several resources to obtain high quality electropherograms. To optimize the efficiency of DNA analysis, we used qPCR as strategy to evaluate PCRs performance before Sanger sequencing reaction and capillary electrophoresis. To test the efficacy of this protocol, real-time PCR was performed using SYBR Safe to amplify 1200 bases of the control region of mtDNA. Real time PCR allowed for detection of successful mtDNA amplification without an agarose gel. Then, the PCR products were purified with AMPURE XP (Beckman Coulter) and eluted in molecular grade water. The sequencing reaction was performed with the BigDye Terminator v3.1 and SYBR Safe (Invitrogen) in a CFX96 Real-Time Thermal Cycler according to the manufacturer’s protocol. A melting curve between 60 and 95 °C was included at the end of the amplification to detect the presence of synthetized DNA fragments. Samples generated peaks of varying intensity at 83 °C on the melting curve, indicating the presence of DNA fragments. The products of the sequencing reaction were purified using CleanSEQ (Beckman Coulter) magnetic beads, and then run in a 3130 Genetic Analyzer (Applied Biosystems). The results demonstrated that samples with peaks higher than 400 RFU on the melting curve generated high intensity electropherograms. Meanwhile, samples with peaks lower than 200 RFU generated no readable-low intensity electropherograms. This methodology allows for rapid determination of sample quality prior to sanger sequencing reaction and capillary electrophoresis allowing to minimize valuable handwork time and reagents.

560 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P479 - VALIDATION OF A NOVEL MASSIVELY PARALLEL SEQUENCING PLATFORM FOR FORENSIC GENETICS Yicong Wang1, Ning Wang1, Jianqiang Wu1, Yifan Xie1, Guifang Yang1, Lin Yang1, Jinwen Yu1, Yanyan Zhang1, Fang Chen1

1 MGI- BGI-Shenzhen, Application research and development center, Shenzhen, China

In recent years, massively parallel sequencing (MPS) has demonstrated its potential for the analysis of short tandem repeats (STRs) and other various forensic markers to complement the weak- ness of capillary electrophoresis (CE) and to obtain more effective information. Miseq FGxTM from Illumina, Ion PGMTM and Ion S5TM from Life Technologies are the most prevalent MPS-based sequencer available for forensic application. Here we introduce a novel massively parallel sequencing platform called MGISEQ-2000 (MGI Tech Co., Ltd, Shenzhen). It’s based on DNBseqTM technology that applies linear amplification in template formation step, with no error accumulation. We have developed a forensic identification panel for DNA analysis based on MGISEQ-2000, which complies 68 autosomal STR, 54 Y-STR, 37 X-STR, 232 SNP loci and 3 regions of mitochondrial DNA in a single PCR multiplex reaction. Sequencing libraries are conducted by a two-step multiplex PCR and then sequenced on MGISEQ-2000 with single-end 400 cycles. In order to validate the performance of the novel platform for forensic analysis, we conducted a study to compare the STR typing consistency between ABI 3730, ABI 3500, Miseq FGx and MGISEQ-2000. The results of this study suggest that MGISEQ-2000 could be used in forensic application, showing very high consistency with conventional CE platforms and Miseq FGxTM platform in both length and sequence polymorphism of STRs. This presentation will focus on the performance of MGISEQ-2000 on forensic genetics and the data comparison of the four DNA typing platforms.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 561 P480 - VALIDATION OF A STREAMLINED RT-qPCR METHOD FOR BODY FLUID IDENTIFICATION David Moore1, Jim Thomson1, Clayton Tim2, Finlay Kennedy3, Daniel Beaumont4

1 Eurofins Forensic Services, DNA Research and Development, Teddington, United Kingdom 2 Eurofins Forensic Services, Casework - Examination and Reporting, Wakefield, United Kingdom 3 Eurofins Forensic Services, Casework - Examination and Reporting, Risley, United Kingdom 4 Eurofins Forensic Services, Casework - Examination and Reporting, Culham, United Kingdom

Forensic DNA STR profiling provides information on the sub-source of DNA samples. However, other source information such as the type of body fluid from which a DNA profile is derived is required to better attribute a matching DNA profile to an individual and to provide activity level evidence. A range of biological molecules such as protein, mRNA and miRNA have been previously used for body fluid identification (BFID). However, many of the published options are complex and require multiple profiling pathways or multiple replicates to obtain interpretable data. We have developed a simplified workflow to deliver an RT-qPCR based system for identification of body fluids using mRNA. The process uses a modified DNA extraction protocol to optimise co-extraction of mRNA and DNA into the same sample eluate, avoiding the requirement for a specialised extraction protocol for mRNA. To simplify the subsequent analysis, we focus on binary tests for two body fluids only, which reflect common questions relevant to different casework scenarios, for example “Does this sample contain saliva or vaginal fluid?” This approach enables the development of smaller, simpler multiplexes using highly specific one-step RT-qPCR assays, designed not to be affected by the presence of genomic DNA. These RT-qPCR multiplexes provide a large dynamic range limiting the number of replicate amplifications required. Here we detail the validation of this streamlined BFID system for identification of saliva and vaginal fluid samples which are commonly encountered in investigations of sexual offences. The study included an assessment on a wide range of sample types obtained from consenting volunteers following sexual activity, providing a robust data set demonstrating the performance of the system.

562 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P481 - VALIDATION OF THE PRECISION ID WHOLE mtDNA GENOME PANEL IN A WORLDWIDE LINEAGE STUDY Christina Strobl1, Jennifer Churchill Cihlar2, Robert Lagacé3, Sharon Wootton3, Chantal Roth3, Nicole Huber1, Lisa Schnaller1, Bettina Zimmermann1, Gabriela Huber1, Harald Niederstätter1, Martin Bodner1, Bruce Budowle2, Walther Parson4,5

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Center for Human Identification, University of North Texas Health Science Center, Texas, USA 3 Human Identification Group, ThermoFisher Scientific, San Francisco, USA 4 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, USA 5 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

As part of the developmental validation study of the Precision ID Whole mtDNA Genome Panel, the performance of the assay was assessed using a total of 526 samples from 24 different worldwide populations. The present lineage study was carried out independently by two laboratories with identical running parameters to evaluate the performance of the assay using samples with various phylogenetic backgrounds. Also, different tissues and quantities/qualities of mtDNA were included. The library preparation for all samples was performed manually and pooled in sets of 32 and 64 samples per chip. This step was followed by automated template preparation using the Ion Chef and sequencing on the Ion S5 System. Raw data analysis was performed using the Ion Torrent Server analysis pipeline and a customized version of the IGV software with parameters specifically developed for the Converge analysis platform. Different parameters were taken into consideration for this study. The concordance between Sanger-type sequencing results and S5 data was assessed for a subset of 251 samples and concordance to Illumina sequencing for 27 samples. Full mitogenome haplogroup estimations were performed for the whole dataset and for the control region and its hypervariable regions and contrasted to haplogroup results using different haplogrouping concepts. Point and length heteroplasmies were evaluated and compared between technologies. The performance of both primer pools was determined through amplicon coverage analysis. Finally, the influence of the number of samples pooled on 530 chips was assessed by performing a base coverage analysis on sequencing runs obtained from chips loaded with 32 and 64 samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 563 P482 - VALIDATION OF THE QUANTIPLEX PRO RGQ DNA KIT Tomasz Kupiec1, Andrzej Doniec1,2, Miłosz Januła1

The measurement of DNA concentration is a very important stage in the process of biological sample analysis for the needs of forensic genetics. At this stage, information is obtained on the quantity and quality of DNA, and the next stages of analysis are planned on the basis of this information. The introduction of the new DNA measurement kit into practice must be preceded by an internal validation process to determine parameters such as sensitivity, linearity, range covered by the method, and intralaboratory repeatability and reproducibility. This publication presents the results of Quantiplex Pro RGQ DNA kit validation experiments using a dedicated Rotor-Gene Q instrument. The reproducibility and repeatability of measurements over a wide range of DNA concentrations were tested in SWGDAM-compliant validation studies. An important element of the study was to determine the sensitivity of the kit to increasing concentrations of inhibitors present in the tested samples. On the basis of DNA samples of varying degrees of degradation, the correlation between the degradation index of autosomal markers and the degradation index for Y chromosome markers was checked. The obtained data were compared with the results of sample amplification with the use of Y-filer Plus and the In- vestigator ESSplex SE QS Kit. In the study it was ascertained that the Quantiplex Pro RGQ DNA kit has an appropriate working range of DNA concentrations for application in practice, and a unique ability to independently determine the degradation index for Y chromosome markers and the degradation index for autosomal markers. The obtained data correlate with the possibility of determining STR markers separately for autosomes and those located on the Y chromosome.

564 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P483 - VALIDATION STUDY AND FORENSIC APPLICATION OF INVESTIGATOR 24PLEX QS KIT Gloria Brescia1, Venusia Cortellini1, Heitor Simoes Dutra Correa1, Nicoletta Cerri1, Andrea Verzeletti1

1 University of Brescia - Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Institute of Legal Medicine, Brescia, Italy

The Investigator 24plex QS Kit (Qiagen) is used for multiplex PCR in forensic human identity and paternity testing. The PCR simultaneously amplifies 22 polymorphic STR markers: the Combined DNA Index System (CODIS) core loci, the European Standard Set (ESS) loci, SE33, DYS391 and Amelogenin. The Investigator 24plex QS Kit is specifically designed for rapid and reliable generation of DNA profiles from blood, buccal swabs, and forensic stains. The kit utilizes QIAGEN’s fast-cycling PCR technology, allowing amplification in around 60 minutes and provides highly robust results with inhibitor-resistant chemistry. The Investigator 24plex QS Kit Primer Mix contains two innovative internal PCR controls (Quality Sensor QS1 and QS2), which are amplified simultaneously with the polymorphic STR markers, to yield helpful information about the efficiency of the PCR and the presence of PCR inhibitors. The Investigator 24plex QS Kit User Guide describes instrument setup and sample preparation of PCR products using the Applied Biosystems 3500 Series Data Collection Software and the Gen- eMapper ID-X Software, but an internal validation is always needed. In this study, we report the successful analysis of amplicons obtained with Investigator 24plex QS Kit on the 3500 Genetic Analyzer. We performed internal validation studies following the Eu- ropean Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP) guidelines, testing several critical areas of kit performance such as sensibility, sensitivity, DNA mixtures and inhibited samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 565 P484 - VERIFICATION OF THE GNANO 31-PLEX ANCESTRY PREDICTION ASSAY FOR FORENSIC CASEWORK Julianne Henry1,2, Catherine Hopkins2, Kelly Hill1, Duncan Taylor1,2

1 Forensic Science SA, Biology, Adelaide, Australia 2 Flinders University of South Australia, College of Science and Engineering, Adelaide, Australia

The Global AIMS Nano (GNANO) 31-Plex SNaPshot assay can be used for biogeographical ancestry (BGA) prediction as it is based on the most differentiated markers of the EUROFORGEN Global AIMS-SNP set and can successfully differentiate five main continental populations; African, Eu- ropean, East Asian, Oceanian and Native American. This system is ideal for implementation into operational forensic DNA laboratories as the test is simple to perform, relatively inexpensive, and can be established on current capillary electrophoresis platforms. Forensic Science SA evaluated the GNANO assay as a non-MPS based option for BGA prediction in forensic DNA casework. This involved assessing parameters such as optimal DNA input amount, analytical threshold, sensitivity, inter- and intra-locus peak balance, artefacts, performance with samples of known ancestry, and performance with degraded DNA. The GNANO assay was shown to be highly sensitive and accurate in regards to BGA prediction, even when a significant proportion of the profile was absent through degradation. However, the presence of SNaPshot artefacts, which mimicked true alleles in negative controls and low level samples, in conjunction with some instances of intra-locus peak imbalance which impacted confidence in calling genotypes, meant that specific interpretation guidelines had to be developed for reporting the results of GNANO. The results of our verification experiments and interpretation guidelines leading to successful implementation of GNANO at Forensic Science SA will be discussed.

566 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P485 - VISUALISING LATENT DNA ON TAPES Piyamas Kanokwongnuwut1, Paul Kirkbride1, Adrian Linacre1

1 Flinders University, College of Science and Engineering, Adelaide, Australia

The collection of latent DNA on items by swabbing or tape-lifting is based on an assumption as to where a person most likely made a contact. Diamond™ nucleic acid dye (DD) has been reported as highly effective at detecting the presence of latent DNA in a range of biological samples (e.g. hair, saliva, fingermarks and skin flakes) and also latent DNA on swabs. This study reports a simple method for visualising and screening latent DNA on tape lifts using a one-step DD staining process followed by visualisation using a portable fluorescence microscope, the “Dino-lite”. A range of tapes was tested, covering tapes currently used by forensic labs for tape-lifting. A range of persons of varying shedder status made contact with items such as fabric, matchstick and paper, for 15 seconds with medium pressure. Tape-lifts were taken from the area of contact and DD applied to the tape. Within 10 seconds stained cellular material was visible under the digital microscope and the number of cells counted. The location of cell staining was removed and direct STR typing performed using Identifiler Plus. This simple visualising technique on tape-lifs allows the cell location to be recorded, and only the area of tape with counted cells removed for DNA typing. The process is a simple and effective triage procedure that reduces the processing of tape samples where there are no cells present.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 567 P486 - WHEN AUTOMATION IS AT ISSUE: DEVELOPMENT OF DNAxs, A SOFTWARE EXPERT SYSTEM FOR DNA PROFILE INTERPRETATION AND MIXTURE ANALYSIS Jord Nagel1

1 Netherlands Forensic Institute NFI, Biological Traces, the Hague, Netherlands

Over the years more loci are used in forensic DNA-profiling to generate more genetic data and therefor increase the discriminating power in casework. This, combined with improved chemistry and increased dynamic range of detection, has however led to more and more complex data analysis. As a consequence the DNA profile interpretation and comparison has become more complex, time-consuming and error-prone. In an attempt to cope with the increased complexity often an array of different programs and ad hoc solutions has been adopted by DNA-scientists. At issue is how integrate these different solutions into one expert system. The Netherlands Fo- rensic Institute (NFI) has therefore develop an integrated software expert system, DNAxs. DNAxs allows DNA-scientists, case data management, within case matching of profiles and complex DNA-profile interpretation using LoCIM inference and computation of consensus and composite profiles. The software has integrated links to other software tools such as SmartRank, Bonaparte and FDStools and CODIS. Within DNAxs the DNAStatistX module is integrated, to effortlessly calculate the evidential value with probabilistic DNA-statistics. This module is based on the continuous maximum likelihood ratio model from EuroForMix. The DNAxs platform aids the DNA-scientists in casework by giving overview and managing the increasingly complex data interpretation and decision making process. In addition DNAxs has increased consistency and accountability, while reducing errors and interpretation variability. The presentation will give insight in the development of DNAxs as an expert system and case management suite and will outline future improvements. An overview of the integrated NFI workflow and efficiency gain with case examples will be demonstrated.

568 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P487 - WHO DO YOU THINK YOU ARE? LONG RANGE FAMILIAL SEARCHES USING THE 23ANDME and GEDMATCH RELATIVE MATCHING TOOLS Christopher Phillips1

1 Institute of Forensic Sciences- University of Santiago de Compostela, Forensic Genetics Unit, Santiago de Compostela, Spain

Much has been made in the last year of so-called “long range familial searching” to generate investigative leads or identify missing persons. This technique applies genetic genealogy tools to genome-wide SNP data obtained from forensic DNA samples, with the aim of identifying a kinship group and then to triangulate a small number of individuals in this often extensive family tree that best match the DNA donor’s likely background. In this study, the author tested his DNA with 23andMe health and ancestry services and used the reported SNP data to explore the closest relative matches. A large number of relatives were discovered using 23andMe’s own tools which were then systematically compared to the cross-genetic-test Genesis toolbox of the GEDmatch community SNP database. The results revealed some surprises and highlighted many of the limitations that are likely to hinder widespread use of such DNA analysis techniques for criminal investigations. In particular, it is clear that the traditional detailed and lengthy genealogical analyses based on public records will continue to be essential in successfully piecing together a kinship group from partial data.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 569 P488 - WHOLE GENOME AMPLIFICATION VERSUS LOW‑TEMPLATE DNA PROFILING: A COMPARATIVE STUDY Hong Han Lim1, Korapin Srisiri1, Budsaba Rerkamnuaychoke2, Achirapa Bandhaya1

1 Faculty of Science- Mahidol University, Forensic Science Unit, Bangkok, Thailand 2 Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Human Genetics Unit, Department of Pathology, Bangkok, Thailand

STR analysis of trace amounts of DNA evidence continues to be challenging, and there is still a need to develop methods to analyse low-template DNA with enhanced quality of STR profiles. Although increasing the number of PCR cycles improves the sensitivity of STR analysis, the reliability of the STR profile is often compromised. Whole Genome Amplification (WGA) has been successfully employed to improve the outcome of subsequent STR analysis by increasing the number of available genome copies. However, there is limited information on how WGA performs in comparison to elevated PCR cycles. This study aims to evaluate the STR profile quality obtained from low-template DNA which had undergone Multiple Displacement Amplifica- tion-type WGA and then amplified by the standard 28-cycle PCR, against that obtained from the low-template DNA typing protocol. Buccal cell DNA samples (10 and 25 pg) were subjected to 16-hour WGA using the REPLI-g® Single Cell Kit followed by 28 cycles of STR amplification using the AmpFLSTR™ Identifiler™ Plus PCR Kit. The stochastic effects observed on the profiles generated were then compared to those from 32-cycle STR amplification. The results showed that, by optimising the dilution factor for the WGA products, artefacts commonly associated with low-template DNA profiling were minimized. The WGA-amplified samples consistently yielded fewer allelic and locus drop-outs compared to samples that were amplified with the 32-cycle PCR. However, the STR profiles from WGA products also resulted in more allelic drop-ins and heterozygous imbalance. The findings suggest that WGA may be an alternative or complementary technique to analyse low-template DNA when sensitized amplification fail to produce conclusive profiles.

570 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P489 - WHOLE GENOME MITOCHONDRIAL DNA ANALYSIS USING MASSIVELY PARALLEL SEQUENCING SYSTEMS Jeff Shaw1, Spencer Hermanson1, Margaret Ewing1, Robert McLaren1, Lotte Downey2, Douglas Storts1

1 Promega Corporation, Research and Development, Madison, USA 2 Promega Corporation, Genetic Identity, Madison, USA

Human mitochondrial DNA is a circular genome, ~16569 base pairs although the number of bases may vary due to small insertions or deletions. Each mitochondrion can have 3–10 copies of mtDNA. For the most part, there are hundreds of copies of mtDNA per cell. In cases where evidence was exposed to harsh environmental elements that can degrade DNA, the high copy number of mtDNA improves the chances of obtaining a DNA result. Massively parallel sequencing simplifies the mitochondrial DNA analysis workflow and provides an opportunity for high-throughput sample processing. Increased mixture deconvolution and heteroplasmy resolution are achieved by deep sequencing coverage and digital read counts, compared to traditional sequencing methods. Additionally, the use of small amplicons to sequence the mitochondrial control region improves sequencing results from degraded samples. The PowerSeqTM Whole Mito System is designed to enable analysis of the entire human mitochondrial genome using MPS. It includes reagents for amplification of the entire 16,569 bp mitochondrial DNA sequence in a single multiplex reaction, generating 161 small amplicons. The use of small amplicons (167 bp average) allows for more robust analysis of degraded samples where nuclear DNA may be of insufficient quantity or quality. Here we demonstrate that the PowerSeqTM Whole Mito System is both accurate and sensitive, giving full mitochondrial DNA profiles with as little as 60 pg of input DNA. In addition, mixture data may be shown.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 571 P490 - YFILER™ PLUS AMPLIFICATION KIT: RAPIDLY MUTATING Y-STR MARKERS AND THEIR PRACTICAL USE IN FORENSICS Petra Pacholíková1, Monika Buresova2, Lukas Vapenik2

1 Police Czech Republic- Regional Police Headquarters Usti nad Labem, Department of Genetic Expertizes, Ústí nad Labem, Czech Republic 2 Police Czech Republic- Regional Police Headquarters Usti nad Labem, Department of Genetic Expertizes, Usti nad Labem, Czech Republic

In our laboratory we analyzed 38 buccal swab samples from 10 groups of paternaly related men using the Applied Biosystems™ Y-STR amplification kit Yfiler™ Plus. The aim of the testing was to check the level of ability of this kit to distinguish men individuals based on the Y-STR profile. Using the standard protocol described in the user manual of the Yfiler™ Plus kit we detected 1 allele mutation in 13 % of the tested samples. The allele mutation was always related to the original allele observed in the Y-STR profile of the oldest person in each group of male relatives. Allele mutations were detected in the loci YGATA H4, DYS576, DYS627, DYS390 and DYF387S1 mainly between the individuals with the largest intergenerational shift. The results of this experiment which will be presented helped us to get an idea about the possible use of the Yfiler™ Plus kit in the forensic practice and its discrimination power. Use of this 27 Y-STR marker kit with 7 rapidly mutating markers seems to be the most efficient tool for routine forensic analysis of Y-STR markers in comparison with other commercially available kits. This type of analysis is often the last option in detecting perpetrators of especially sexual abuse crime in cases there are paternally related individuals. This kit can be also used for obtaining at least the operative information for law enforcement responsible for investigation of serious type of crime in cases when we are not able to analyze the autosomal STR profiles due to the low template or degraded DNA. This is possible also thanks to the 11 Y-STR loci in mini-STR format (below 220bp) present in the Yfiler™ Plus kit.

572 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS FORENSIC MATHEMATICS AND STATISTICS

P491 - A DEEP LEARNING APPROACH FOR PAIRWISE KINSHIP INFERENCE Lu Yin1, Weibo Liang1, Jing Zhu1, Shengqiu Qu1, Yinji Wang1, Lin Zhang1, Meili Lv2, Sicheng Huang3

1 Sichuan University, Department of Forensic Genetics West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Sichuan University, Department of Immunology West China School of Basic Medical Sciences and Forensic Medicine, Chengdu, China 3 Chengdu Public Security Bureau, Department of science and technology division, Institute of criminal investigation, Chengdu, China

Kinship inference is an important task in forensic genetics, and the development of High-Through- put Sequencing technologies has given researchers a mass of information from which can infer familial relationships. Up to now most kinship analysis approaches usually use methods based on Markov chain, Bayes’ theorem and likelihood calculations. They often focus on a small part of features of the data. So that it is possible to miss useful information in the data. Unlike these methods, deep learning does not require predefined features but discovers meaningful features from our labeled training data so as to make inferences[1]. In this study, we developed a deep learning method to inference pairwise kinship make use of single nucleotide polymorphism (SNP) profiles in the whole genomics. We convert the alignment of segregating SNP sites of two individuals into images and construct a convolutional neural network to predict pairwise familial relationships. Our CNN model contains a standard feedforward neural network and a 2D convolutional neural network with a fully connected layer. We chose some suitable SNP loci and numbers of pairwise relationships to train and evaluate the neural network. The model is still in the training step. The follow-up work is ongoing and we hope for good results. [1] L. Flagel, Y. Brandvain, D.R. Schrider, The Unreasonable Effectiveness of Convolutional Neural Networks in Population Genetic Inference, Mol Biol Evol (2018).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 573 P492 - A FORENSIC MARKER SELECTION METHOD FOR POPULATION STRUCTURE EVALUATION Yuxiang Zhou1, Yining Yao1, Baonian Liu1, Qinrui Yang1, Kuan Sun1, Chengchen Shao1, Qiqun Tang2, Jianhui Xie1

1 Fudan University, Department of Forensic Medicine, School of Basic Medical Sciences, Shanghai, China 2 Fudan University, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai, China

In the previous study, we found that with an increasing number of Y-STRs in haplotype sets, genetic distances between populations decrease or increase, demonstrating that each locus has different genetic diversities among populations. Thus, we aimed to develop a method to obtain markers with optimal genetic diversity. A novel marker selection approach that measures the average genetic variance of single locus in a collection of populations was proposed. It is defined as PSIndex (Population Structure Index) =1/n Σ n j

574 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P493 - A MULTIVARIATE STATISTICAL TOOL FOR THE EVALUATION OF THE BIOGEOGRAPHICAL ANCESTRY INFORMATION FROM TRADITIONAL STR DATA Eugenio Alladio1, Chiara Della Rocca2, Fulvio Cruciani2, Marco Vincenti3, Paolo Garofano4, Filippo Barni1, Andrea Berti1

1 Reparto Carabinieri Investigazioni Scientifiche di Roma, Sezione di Biologia, Roma, Italy 2 Sapienza Università di Roma, Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Roma, Italy 3 Università degli Studi di Torino, Dipartimento di Chimica, Torino, Italy 4 Centro Regionale Antidoping e di Tossicologia “A. Bertinaria” di Orbassano Torino, Laboratorio di Biologia e Genetica Forense, Orbassano Torino, Italy

The interpretation of data from DNA profile genotyping is one of the most relevant topics in forensic science; among other applications, genetic profile’s capability to distinguish ethnic groups and biogeographic ancestry (BGA) affiliations have been largely explored. In fact, for investigative and security purposes, it would be extremely useful to identify subjects and estimate their origins just by examining traditional STR DNA profiles. Current protocols for ethnic origin estimation using STR profiles are usually based on Principal Component Analysis approaches and Bayesian methods. The present study provides an alternative and dynamic tool that involves the use of several multivariate data analysis strategies for estimation of the BGA information from unknown biological traces. Powerful multivariate techniques such as, for instance, Partial Least Squares-Discriminant Analysis (PLS-DA) and Support Vector Machines (SVM) are employed and their discriminating power has been compared. These techniques have been applied on specific population datasets containing the allele frequencies of several African populations and tribes, Caucasian individuals and Asian subjects. Both PLS-DA and SVM techniques provided robust classifications, yielding high sensitivity and specificity models capable of discriminating the populations on ethnic basis. Lastly, examples of real cases have been examined, and the developed models have been easily extended to smaller and more specific populations. The developed interpretation approach is aimed to be converted into an open-source user-friendly R Shiny app in order to represent a helpful and powerful instrument for law enforcement agencies and analysts.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 575 P494 - A NEW STRATEGY TO IDENTIFY FOUR KINDS OF BODY FLUIDS USING 6 miRNAs Sun Qifan1, Yuan Li2, Ji Chengjie3, Jiang Li1, Zhao Yixia1, Li Ranran1, Hu Sheng1, Li Caixia1, Wang Le1, Ye Jian1, Ji Anquan1

1 institute of forensic science, Innovation and Development Research department, Beijing, China 2 Shandong University, Mathematics college, Qingdao, China 3 Sichuan Provincial People’s Hospital, Clinical Laboratory, Chengdu, China

Abstract: Body fluids (or their stains) identification plays important role in reconstructing the crime scene or providing evidence in court. MicroRNA (miRNA) is a kind of molecular with small size and tissue specificity, and also can be resistant to degradation in extreme environments. The analysis of specific miRNAs using RT-qPCR has been proposed to be an effective and sensitive approach to perform the body fluids identification test. Peripheral blood, menstrual blood, saliva and semen are common body fluids exist in crime scene. In our study, 6 kinds of specific miRNAs reported by published papers and a housekeeping gene RNU6b were selected and the relative expression quantity of these miRNAs were detected by real-time PCR, with 105 samples from 5 different kinds of body fluids, including peripheral blood samples, menstrual blood samples, saliva samples and semen samples. The expression patterns of the 6 miRNAs were completely different in the 4 kinds of body fluid and the analysis result were shown by columnar graph, heatmap and PCA. Based on these expression data, a computational algorithm using the Soft Max regression model was successfully established to evaluate the likelihood of the certain type of each unknown body fluids sample. Using the developed model, we got a 100 % accuracy result when determine the aiming body fluids in a set of test samples. Key words: Body fluids identification, miRNA, real-time PCR, Soft Max regression model, likelihood.

576 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P495 - APPLICATION OF A METHODOLOGY TO EVALUATE DNA RESULTS WITH PROPOSITIONS AT THE ACTIVITY LEVEL USING BAYESIAN NETWORK Lydie Samie1, Christophe Champod1, Franco Taroni1

1 University of Lausanne, School of Criminal Justice, Lausanne, Switzerland

It is fair to say that many experts are not at ease considering their results associated with traces of low level of DNA in the light of alleged activities. One of the reasons is that each case has its own set of features and it is difficult to design experiments taking into account all of them. However, the real issue is not revolving around the number of influencing factors but more on which of them has a significant impact on the Likelihood Ratio. Firstly, we will present a methodology that allows to identify the most impacting variables on the LR. This methodology takes advantage of Bayesian networks coupled with simulations techniques to identify these variables. It allows then to focus the acquisition of data on these variables only. Secondly, we applied the methodology on simulated stabbing cases. From these “known ground truth” cases, potential DNA recovered from the knife handle is assessed against a given set of propositions, namely that the person of interest, the POI, stabbed the victim (primary transfer) or that the POI shook the hand of the real alternative offender, the AO (secondary transfer). Two couples of different DNA shedders are used. For each alleged activity and for each couple, 30 experiments reproducing the stabbing scenario have been performed and the associated DNA results have been evaluated. Results show that if the POI is a poor DNA shedder compared to the alleged AO, the observations support the correct proposition over the alternative in all cases except one. However, if the POI is a better DNA shedder than the alleged AO, the observations support the correct proposition against the alternative in all 30 cases when the activity involved the primary transfer and only 14 out of 28 cases when the activity involved the secondary transfer.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 577 P496 - APPLYING AUTOSOMAL STR PROBABILISTIC GENOTYPING MODELS TO SNP DATA USING HIERARCHICAL BAYESIAN MODELLING Duncan Taylor1, Julianne Henry1, Catherine Hopkins1, James Curran2

1 Forensic Science SA, Biology, Adealide, Australia 2 The University of Auckland, Statistics, Auckland, New Zealand

The global AIMS Nano (GNano) profiling system is a set of 31 SNPs that has been shown to perform well at differentiating individuals from the five continental population groups of Africa, Europe, East Asia, Native America and Oceania. Recently at Forensic Science SA we investigated the performance of the GNano system on Aboriginal Australians, with a view to implementing the GNano system as an investigative service. As well as the validation of the ancestry assignment, an assessment of the DNA profile stochastic behaviour was carried out. The purpose of the assessment was to develop interpretation guidelines that can be used when a GNano profile is generated from an evidence sample under non-optimal conditions (i.e. low levels of DNA, degraded DNA, or a mixed DNA sample). To model DNA profile behaviour, Hierarchical Bayesian Modelling was carried out, trialing different combinations of profile parameters and model architectures (based on analogous models in autosomal STR systems). The optimal model, based on model comparators, was chosen and used to develop interpretation guidelines (as an interim measure until the models can be applied directly in a probabilistic genotyping system). The result of this work is the implementation of GNano at Forensic Science SA in active casework as a tool to assist investigations that are otherwise exhausted.

578 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P497 - BAYESIAN NETWORKS FOR EVALUATING SNP-STR PROFILING RESULTS FROM UNBALANCED DNA MIXTURES Hui Jian1, Li Wang2, Yu Tan1, Ranran Zhang Zhang1, Yuqing Liu1, Weibo Liang1, Lin Zhang1, Meili Lv3

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Sichuan University, Department of Obstetrics and Gynecology, West China Second University Hospital, Chengdu, China 3 Sichuan University, Department of Immunology, West China School of Basic Medical Sciences and Forensic Medicine, Chengdu, China

SNP-STR is a compound genetic marker composed of a SNP locus and a closely linked STR, and it has been confirmed that the SNP-STR haplotypes of minor component of DNA mixtures with a ratio of 1:500 could be specifically amplified using amplification refractory mutation system (ARMS) technic in our previous study1, 2. 18 SNP-STR loci have been screened and the corresponding population study was carried out in a southwest Chinese Han population in our previous study. Here we developed a novel analytical approach for the probabilistic evaluation of SNP–STR profiling results obtained from unbalanced DNA mixtures. The procedure was based on Bayesian networks and used the likelihood ratio as an expression of the probative value, allowing one to provide a clear description of the genotypic configuration observed from the mixed stain, and perform the necessary probabilistic computations. By using this framework for one casework example, the results showed that SNP-STR could provide robust likelihood ratio values for the reported casework example, while autosomal STRs exhibited limited performance, thus indicating that the method we developed in this study could serve as an alternative method for the analysis of extremely unbalanced two-person DNA mixtures. 1. Wang L., Li Y., Tan Y., et al., SNP-STR analysis for non-invasive paternity test for fetus(2017), Forensic Sci. Int.: Genet. Suppl. Ser. 6: e413-e414. 2. Tan Y., Bai P., Wang L., et al., Two-person DNA mixture interpretation based on a novel set of SNP-STR markers(2018), Forensic Sci Int Genet. 37: 37–45.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 579 P498 - BAYESIAN NETWORKS FOR SOURCE LEVEL ATTRIBUTION OF SALIVA: DEVELOPMENT AND APPLICATION IN CASEWORK Sallyann Harbison1, Tayla Schaapveld2, Stephanie Opperman1

1 Institute of Environmental Science and Research Ltd, Forensic Biology, Auckland, New Zealand 2 University of Auckland, Forensic Science Program- School of Chemical Sciences, Auckland, New Zealand

Bayesian networks and evaluative reporting have been available to forensic scientists for some time and are gaining traction in forensic biology. Forensic DNA profiling provides an evaluation of the evidence at the sub source level according to the hierarchy of propositions. In many court cases, this DNA evidence is accepted and any challenge becomes from what did the DNA originate, how did it get there and when. Before tackling the complexity of activity level propositions (the how), we have developed a Bayesian network for the identification of saliva at the source level in the hierarchy. Here we present our network, show its strengths and limitations, explain how we use it in forensic casework and provide casework examples.

580 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P499 - COMPARISON OF WEIGHT OF EVIDENCE RESULTS GENERATED FROM TWO MCMC BASED PROBABILISTIC GENOTYPING SOFTWARE Megan Foley1, Nicholas Wolford1, Heather Mckiernan1

1 CFSRE, Forensic Biology, Willow Grove, USA

This study compared likelihood ratio results generated from two fully-continuous probabilistic genotyping platforms, BulletProof and GenoProof Mixture 3, that utilize Markov chain Monte Carlo (MCMC) based algorithms. Two-, three-, and four-person mixtures were prepared targeting various input concentrations of total DNA. Overall input amounts ranged 950 pg (major contributor) and 2.5 pg (minor contributor). All mixtures were amplified using the Promega® PowerPlex® Fusion 6C amplification kit and DNA profiles were generated using Applied Biosyste- ms 3500 Genetic Analyzer. Likelihood ratios were calculated a total of five times per contributor on each platform. This test for repeatability was performed to evaluate the run-to-run variation due to random sampling associated with MCMC based methods. Intra– and inter-platform performance was then assessed. True number of contributors were utilized for all hypotheses. A validated drop-in value of 0.01 was used as was a validated scaled probability of dropout. All other software settings were kept as identical as possible. Mixtures that contained higher concentrations of DNA generated more repeatable results within each platform for all contributors. Intra-platform variability was minimal, varying between 1–2 orders of magnitude. More variability in likelihood ratios generated between platforms was observed as compared to within. Over- all, this study demonstrated minimal variability associated with two fully-continuous platforms using MCMC-based sampling resulting in high degrees of repeatability.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 581 P500 - COMPLETION OF THE MIX 13 CASE STUDY BY EVALUATION OF MOCK MIXTURES WITH THE PROBABILISTIC GENOTYPING SOFTWARE GENOPROOF® MIXTURE 3 Maria Harthun1, Aline Jede1, Holger Schönborn2, Anne-Marie Pflugbeil3

1 qualitype GmbH, Scientific Support, Dresden, Germany 2 qualitype GmbH, Software Development, Dresden, Germany 3 qualitype GmbH, Business Development, Dresden, Germany

Only recently, at the end of 2018, the results of the MIX 13 interlaboratory study, initiated by NIST in 2013, were published. Aiming to evaluate current available methods for DNA mixture analysis in the US and Canada, the complex DNA mixtures were reviewed from new perspectives, taking probabilistic genotyping methods into account. Based on the results of this study, the presented scenarios were analysed with the software GenoProof® Mixture 3. For this purpose, the readily accessible sample data was used. Focusing on the evaluation of the results in comparison with predetermined references, LR results were additionally compared between probabilistic software presented in the publication of Buckelt- on et al. Particular attention was paid to the evaluation of hypotheses associated with scenario 5. In this case, the deconvolution of involved persons, considering given hypotheses in the scenario, proved to be difficult. The results obtained with the GenoProof® Mixture 3 software are presented and subsequently discussed in this study.

582 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P501 - DEVELOPMENT OF A NEW SOFTWARE FOR ESTIMATING CONFIDENCE INTERVAL OF STATISTICAL INDEXES USED FOR DNA EVIDENCE INTERPRETATION Sho Manabe1, Chie Morimoto1, Shuntaro Fujimoto1, Eriko Hirai1, Yuya Hamano1, Keiji Tamaki1

1 Kyoto University Graduate School of Medicine, Forensic Medicine, Kyoto, Japan

There are several aspects that influence the uncertainty of the allele frequencies when calculating statistical indices, including random match probability (RMP), combined probability of inclusion (CPI), or likelihood ratio (LR), that are used for interpretation of crime stain profiles. The size of the population database has a significant effect on the allele frequencies, especially on the minimum frequency. The confidence intervals of these frequencies and statistical indices are useful in the consideration of the uncertainty in a finite population database. In this study, new software was developed to estimate the confidence intervals of some statistical indexes used for DNA evidence interpretation. The software has a graphical user interface written in R language (ver. 3.5.2). A Dirichlet distribution is used to consider the uncertainty of allele frequencies in each locus. Based on allele counts of the population database, the software automatically calculates the parameters of the Dirichlet distribution. The values of RMP, CPI, and LR of a binary model can be calculated using the expected allele frequencies based on the Dirichlet distribution. In addition, the variation of these values can be estimated by repeated random sampling of the allele frequencies of the distribution (e.g., 10,000 times). This variation is then used to estimate the confidence interval in a user-selected range. The software also fa- cilitates the estimation of the confidence interval of LR values for pairwise kinship analysis via the same approach. We will present several examples of estimated confidence intervals using commercially available typing kits of short tandem repeats.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 583 P502 - ESTIMATES OF MUTATION RATES FROM INCOMPATIBILITIES ARE MISLEADING - GUIDELINES FOR PUBLICATION AND RETRIEVAL OF MUTATION DATA URGENTLY NEEDED António Amorim1,2,3, Nádia Pinto1,3,4

1 Institute of Pathology and Molecular Immunology from University of Porto IPATIMUP, Population Genetics and Evolution, Porto, Portugal 2 Faculty of Sciences of the University of Porto, Biology, Porto, Portugal 3 Institute for Innovation and Research in Health i3S- University of Porto, Portugal, Population Genetics and Evolution, Porto, Portugal 4 Center of Mathematics of the University of Porto, Porto, Portugal

Unbiased estimation of germinal mutation rates are essential for population and evolutionary genetic analyses and more so in any forensic kinship evaluation. A major source of bias in diploid or haplodiploid loci has been identified – ‘hidden’ or ‘covert’ mutations (i.e. those not causing Mendelian incompatibilities) and ways to overcome it proposed. Furthermore, the identification of (a) the parental meiosis in which mutation has occurred, as well as (b) the allele of origin and the mutated one, are problematic. Again, a pseudo most parsimonious approach goes on being used although it has already been shown that misclassification of mutations has a significant impact, overestimating one-step mutations and underestimating all others. We have confirmed and extended these findings in autosomes to X chromosome markers and derived the formulas for the expected frequencies of duos and trios with possible hidden mutation, showing that for commonly used STRs, mutations are detected with certainty in less than 10 % of the cases. In forensics, current publication and other publicly obtainable data formats ignore both types of bias and do not allow more than the estimation of average incompatibility rates per locus. These values are biased and, in kinship analyses, estimates of biallelic specific mutation rates should be used for correct likelihood calculations. We urge the forensic community to undertake the task of preparing guidelines for archival, retrieval and most specially, publication of data in a format that will allow a more accurate estimation of this crucial parameter, providing a solid ground for the formulation and testing of mutation models. For this, the publication of the complete set of observed genotypes in duos and/or trios in the population sample is mandatory.

584 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P503 - EVALUATION OF MACHINE LEARNING ALGORITHMS FOR IDENTIFICATION OF HUMAN REMAINS IN DVI INCIDENTS Juan Luque1, Esteban Vegas2, Alex Sanchez-Pla3

1 INTCF-Barcelona, Biologia, Barcelona, Spain 2 Barcelona University, Departament of Genetics, Microbiology and Statistics, Statistics Section, Barcelona, Spain 3 Barcelona University, Departament of Genetics, Microbiology and Statistics, Barcelona, Spain

In the context of a disaster victim identification (DVI) incident it is essential to achieve a quick and effective match of human remains with their relatives. In the present work we have explored the possibility of applying machine learning techniques to the resolution of identifications in a DVI incident. In order to apply this type of techniques it is essential to have a large enough training set. For that reason, we have designed a large synthetic family with a pedigree of five generations and 19 individuals and generated short tandem repeats (STR) profiles for all the individuals of 40,000 large families, based on such pedigree. In order to improve the effectiveness of the algorithms, the training set has been transformed in a way that takes into account the match between alleles from a pair of compared individuals, as well as the population frequencies. After evaluating several models, a keras / tensorflow neural network was implemented and trained with R so that it could be used to predict the kinship of samples given the multiple DNA profiles obtained in a DVI. Ten settings of events with multiple victims (from 6 to 200 victims) were simulated, with different levels of difficulty. The inference of the neural network in such settings has solved most of the identifications in a few minutes. In some of the settings, the identifications were solved with a 100 % accuracy, while the most complicated setting with the farthest family relationships could only obtain a 50 % accuracy. In the future, we are planning to improve the method and develop a user-friendly interface so that it can be used in forensics laboratories.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 585 P504 - EVALUATION OF SENSITIVITY AND SPECIFICITY OF SIBSHIP DETERMINATION IN THE CAUCASIAN POPULATION OF THE RUSSIAN FEDERATION USING 23 STR LOCI Vladislav Zavarin1, Viktoriya Ilina1, Yevgeniy Krassotkin1, Tatiana Makarova1, Andrei Semikhodskii1

1 LLC Medical Genomics, Laboratory for Molecular Genetics, Tver, Russian Federation

The 13th edition of AABB Standards for Relationship Testing Laboratories contains new requirement to report “the estimate of the percentage of individuals of known relationship that may have a combined likelihood ratio that is inconclusive, or supportive, or not supportive of the tested relationship” for two-party comparisons of full sibling, half sibling, avuncular, and single grandparent. We performed a simulation study to evaluate sensitivity and specificity of sibship determination in the Caucasian population of the Russian Federation using 23 autosomal STR loci included in the VeriFiler Express kit. Percentage of true and false positives was determined using LR threshold 10. Percentage of true and false negatives was determined using LR threshold 0.1. For half siblings duo: simulating the true relationship, 72.7 % of the 1000 simulations were equal or above the LR limit, 1.7 % of false negatives; simulating the alternative hypothesis yielded 72.8 % of true negatives and 1.0 % of false positives. For half siblings (one mother will be genotyped): simulating the true relationship, 85.3 % of the 1000 simulations were equal or above the LR limit, 0.9 % of false negatives; simulating the alternative hypothesis yielded 87.6 % of true negatives and 0.4 % of false positives. For half siblings (mothers will be genotyped): simulating the true relationship, 93.0 % of the 1000 simulations were equal or above the LR limit, 0.5 % of false negatives; simulating the alternative hypothesis yielded 94.1 % of true negatives and 0.3 % of false positives. For full siblings: simulating the true relationship, 98.6 % of the 1000 simulations were equal or above the LR limit, 0.3 % of false negatives; simulating the alternative hypothesis yielded 98.5 % of true negatives and 0.2 % of false positives.

586 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P505 - EVOLUTIONARY OPERATOR FOR CALCULATING THE FREQUENCY OF OCCURRENCES OF MANY ALLELIC VARIANTS OF THE GENES HLA, STR, AND Y-STR LOCI Sardarxodja Kurganov1, Karim Kurganov2

1 Republican Centre of Forensic Expertise, Laboratory of Forensic Biological Examination of Human DNA, Chilonzor- Tashkent, Uzbekistan 2 National University of Uzbekistan, Department of Mechanics and Mathematics, Vuzgorodok- Tashkent, Uzbekistan

The requirement for compatibility of growing DNA database has led to solutions of problems arising in the field of mathematical genetics, in which quadratic stochastic operators can be used. In this work, we proposed the attempt to simulate the variability of the picture, having many allelic variants of the example of genes HLA, STR, and Y-STR loci. To solve this problem, the Malthus model and the stochastic model constructed by the authors were used. Here, the necessary information from the theory of QSOs is presented. In simplex, S ^ (n-1) = {x = (x_1; x_2;…; x_n): ∑_ (i = 1) ^ n (x_i) = 1, x_i≥0} consider the evolutionary population operator (Vx)_k=x_k^’=∑_(i,j=1)^n P_(ij,k) x_i x_(j,) k=¯(1,n), where, P_(ij,k)≥0, P_(ij,k)=P_(ji,k), ∑_(k=1)^n P_(ij,k) =1,x=(x_1; x_2;…; x_n ) S^(n-1). The conditions put on the coefficients P_(ij,k) ensure the preservation of the simplex〖 S ^ (n-1)〗, i.e. V(S ^ (n-1)) S^(n-1) The state of alleles can be specified〖〗 as a set of x=(x_1^0;…;x_n^0 )∊ probabilities of species. The P_(ij,k) coefficients are the probability of the appearance of the k -allele during the i-mutation⊂ and the j- allele. There- fore, x_k=∑_(ij,k=1)^n P_(ij,k) x_i x_j , k=(1,n) , will be the total probability. If in some generation the alleles are in a state of x, in the next generation it is in a state of x^’=Vx. 〖〗 ̅ If the generation of alleles is in the Malthus model x_k^’=α_k*x_k, where the coefficient α_k is found in the following form α_k=[1-∑_(i=1)^n x_i-x_k )x_i ], k=1,…,n then the evolution operator is written as: V{(x_1^,=x_1 [1-(x_2-x_1 ) x_2-(x_3-x_1 ) x_3-(x_4-x_1 ) x_4-…-(x_n-x_1 ) x_n ], x_2^,=x_2 [1-(x_1-x_2 ) x_1-(x_3-x_2 ) x_3-(x_4-x_2 )〖( x_4-…-(x_n-x_2 )〗 x_n ],...x_n^,=x_n [1-(x_1-x_n ) x_1-(x_2-x_n ) x_2-(x_3-x_n ) x_3-…-(x_(n-1)-x_n ) x_(n-1) ].)}

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 587 P506 - FAMLINK2 – A COMPREHENSIVE TOOL TO ACCOMMODATE GREAT NUMBERS OF LINKED MARKERS ACCOUNTING FOR MUTATIONS AND SUBPOPULATION EFFECTS Daniel Kling1, Andreas Tillmar2

1 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway 2 National Board of Forensic Medicine and Forensic Toxicology, Department of Forensic Genetics, Linköping, Sweden

We present an extension of existing software to perform likelihood calculation in kinship cases. FamLink2 builds on the algorithm presented in previous studies and adds support for subpopulation correction as well as provide a general implementation of likelihood calculations for virtually unlimited linked autosomal markers. We provide an overview of the features and why the software is crucial given the current development in relation to expanded marker panels. We demonstrate an evaluation of some common kinship cases using simulations and illustrate how accounting for linkage will affect the results for some available combinations of marker panels.

588 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P507 - FORENSIC COMPUTATIONS IN PEDIGREES WITH INBRED FOUNDERS Magnus Dehli Vigeland1, Egeland Thore2

1 University of Oslo, Medical Genetics, Oslo, Norway 2 Norwegian University of Life Sciences, Faculty of Chemistry, Biotechnology and Food Science, Aas, Norway

A standard assumption in forensic pedigree analysis is that the pedigree founders are non-inbred. The fact that this is false in most practical applications (since we are all related at some level) is usually silently ignored, in the hope that the “background inbreeding” has a negligble effect. However, as we will show, this hope is not always realistic. The problem of modeling background inbreeding in pedigrees is generally difficult, except for very simple situations where explicit formulas are obtainable. One approach is to extend the pedigree in question by adding extra layers of suitably related ancestors. However, this quickly leads to unwieldy pedigrees usuitable for computer analysis. For example, modeling low levels of inbreeding may require great numbers of extra ancestors in order to construct the necessary distant relationships. Similar challenges occur for individuals with very high levels of inbreeding, as typically encountered with model organisms. We propose an extension of the standard framework for pedigree analysis, by allowing pedigree founders to be inbred. We introduce the R package forrel, offering a range of forensic pedigree calculations and visualisations. A core feature of this package is that any pedigree founder may be assigned an arbitrary inbreeding coefficient, which is respected in all subsequent calculations. As a result it becomes easy to explore the effect of background inbreeding in many forensic computations, including likelihood ratios, exclusion probabilities, simulation studies and pedigree reconstruction. In our talk we will show several examples, both of theoretical and practical interest.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 589 P508 - HYBRIDMARK: ELIMINATING THE PCR HYBRID ARTEFACTS IN FORENSIC MPS DNA PROFILES Jerry Hoogenboom1, Nathan Hoogendorp1, Kristiaan van der Gaag1, Titia Sijen1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

By transitioning from Capillary Electrophoresis (CE) to Massively Parallel Sequencing (MPS), the complete sequence of each DNA fragment is obtained, instead of just the length. Besides an increase in discriminatory power, this also allows for much more accurate detection and filtering of artefact products that have been generated during amplification and sequencing. The presence of artefacts limits the ability to identify the alleles of a minor contributor in a mixture. PCR stutter is the predominant type of artefacts with Short Tandem Repeat (STR) analysis. Stutter artefacts can be filtered both in CE profiles and MPS data; the filtering in MPS data is more accurate since not only the allele length but also the allele sequence is considered. However, MPS unveils an amplification artefact generally not noted in CE analysis namely PCR hybrids. The sequences of these artefacts show a combination of two alleles. These PCR hybrids are the predominant type of artefact after the stutter artefacts. Since they can reach up to 10 % of the reads at an STR locus, they could be mistaken for a low-level contribution. PCR hybrids are often difficult to recognise by the human eye, requiring careful comparison of the various sequences detected within a sample. Therefore, we introduce HybridMark to automatically identify sequences that may represent PCR hybrids. Since HybridMark is added as a feature in the FD- STools software package, FDSTools can now not only apply noise correction for STR stutters, but also efficiently eliminate PCR hybrid artefacts. This makes interpretation of mixed MPS DNA profiles manageable even regarding very minor contributions such as 1 in 40.

590 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P509 - INTEGRATING CONCEPT FOR GENETIC IDENTITY ASSESSMENT Irina Perepechina1

1 Lomonosov Moscow State University, Department of Criminalistics of the Faculty of Law, Moscow, Russian Federation

Here we consider a holistic, integrating concept of the genetic identity assessment, which brings together and further developes our previous research in this field, including two different approaches which were presented at the 27thCongress of the ISFG in Seoul (Perepechina, 2017). Complex consideration provides a comprehensive look at the problem. The general idea is that the level of reliability of forensic identification should be set by the requirements of the society (scientific community, law enforcement system, public). The key element of the concept is empirically adopting the level of type I vs type II judgment errors by the society, which needs to be elicited from special polls. Despite each case is unique, we argue that it is fundamental- ly possible to define a scientifically grounded identity threshold and accept it as a standard. The standard is to be developed on the basis of the predetermined high consensual level of reliability, justified by the society and meeting the requirements of the national judicial system. As a technology of choosing the standard, analyzing a series of the equivalent probabilities is proposed that provides for multilaterally assessing the risk of the identification error. Along with the assessing the objective statistical value of reliability of DNA identification considered above, the problem also includes the aspect related to the subjective factor - the adoption of the decision regarding the identity threshold. The developed mathematical model employs the hypothesis of the rational behavior to reveal the factors influencing decision-making, and to study the border probability values (Goubko and Perepechina 2015). The concept seems to be applicable not only in DNA analysis, but also in other areas of forensic identification.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 591 P510 - INTERPRETING MULTIPLE LR VALUES FROM DIFFERENT PROBABILISTIC SOFTWARE: A JOINED APPROACH INVOLVING LOGISTIC REGRESSION Eugenio Alladio1, Chiara Della Rocca2, Monica Omedei3, Selena Cisana4, Denise Caneparo3, Filippo Barni5, Andrea Berti5, Tereza Neocleous6, Paolo Garofano3

1 Università degli Studi di Torino, Dipartimento di Chimica, Torino, Italy 2 Sapienza Università di Roma, Dipartimento di Biologia e Biotecnologie “C. Darwin”, Roma, Italy 3 Centro Regionale Antidoping e di Tossicologia “A. Bertinaria” di Orbassano Torino, Laboratorio di Biologia e Genetica Forense, Orbassano Torino, Italy 4 Centro Regionale Antidoping e di Tossicologia “A. Bertinaria” di Orbassano Torino, Centro Regionale Antidoping e di Tossicologia “A. Bertinaria” di Orbassano Torino, Orbassano Torino, Italy 5 Reparto Carabinieri Investigazioni Scientifiche, Sezione di Biologia, Roma, Italy 6 University of Glasgow, School of Mathematics and Statistics, Glasgow, United Kingdom

Complex STRs profiles such as DNA mixtures obtained from crime scene evidence still represent one of the most demanding and time-consuming challenge for DNA analysts. Their interpretation may turn to be very problematic, particularly in case of external factors occurrence such as random sample degradation and contamination Moreover, another source of complexity is the fact that there are three models, with different complexities’ degrees, which can be used for probabilistic genotyping: (i) the binary approach (actually obsolete), (ii) the semicontinuous approach and (iii) the continuous-quantitative approach. However, despite the Forensic Commu- nity has been proposing several recommendations over the past few years, a unique rigorous LT-DNA mixture interpretation procedurehas still to be defined. For these reasons, the main goal of this study has been the development of a comprehensive application capable to combine the likelihood ratios’ (LR) results from different probabilistic software. For this purpose, several 2-, 3- and 4-person mixtures have been set up, analyzed and interpreted by means of different probabilistic software involving both the semicontinuous (LRmix Studio and Lab Retriever) and the continuous (DNA•VIEW®-Mixture Solution, EuroForMix and STRmixTM) approach. Logistic regression models have been proposed to combine the different LR values, together with the degradation index and DNA quantification information. Furthermore, a dynamic, user-friendly and open-source R Shiny application has been developed to allow analysts to deal with these LR data. The application software provides a unique LR value that eases the interpretation process and might represent a valuable approach to be shown in courtrooms, especially in case of contradictory LR results.

592 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P511 - IS LINKAGE SIGNIFICANT IN COMPLEX KINSHIP TESTING? A PRELIMINARY SIMULATING STUDY BASED ON THE GOLDENEYE™ 20A KIT Yinming Zhang1,2, Haixia Li1,2, Nana Wang1,2, Xuefeng Shen1,2, Hongyu Sun1,2

1 Faculty of Forensic Medicine- Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China 2 Guangdong Province Translational Forensic Medicine Engineering Technology Research Center, Sun Yat-sen University, Guangzhou, China

Recently, the genotyping systems containing more STR loci have been established and used in forensic applications, thus increasing the possibility of including the loci at the same chromosome in one system, which makes the problems of linkage and recombination events should be considered. Lots of researchers have concluded that the difference of likelihood ratio calculated ignoring or considering linkage in kinship testing could be significant. Meanwhile, other studies also have demonstrated that in some circumstances, linkage considerations on the LR value of two syntenic loci might still not challenge the conclusions. In this study, we selected Gold- enEyeTM 20A kit as a templating system to evaluate the effect of linkage in kinship testing, which is one of the most widely used systems in China and contains three pairs of markers located at the same chromosome respectively (CSF1PO and TPOX, D12S391 and vWA, D21S11 and Penta D). Firstly we summarized the formulas of the LRs based on the recombination fraction (r value) and genotypes of two linked STR loci of two individuals. Then we simulated 10,000 cases of sibling, grandparent-grandson and uncle-nephew relationships, respectively. Based on the formulas and simulated genotypes, we obtained the distribution of the LR value with the consideration of linkage (exact values). Compared with the values taken all the loci as independent ones (rough values), the differences between the exact and rough values were not significant. We suppose that for the GoldenEyeTM 20A kit, ignoring the linkage is acceptable in sibling, grandparent-grandson and uncle-nephew testings.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 593 P512 - ISOALLELIC FREQUENCY ESTIMATION FOR STR MARKERS FROM MASSIVE PARALLEL SEQUENCING DATA Torben Tvedebrink1,2, Mikkel Meyer Andersen1,2

Massive parallel sequencing (MPS) continues to receive increasing interest in the forensic community. The MPS technology allows more markers (short tandem repeat, STR, and single nucleotide polymorphism, SNP) to be genotyped simultaneously, including markers for ancestry investigations (AIMs), phenotypic trait predictions and lineage analysis (Y-chromosomal markers, SNPs and STRs). In addition to the enlarged set of markers genotyped from the same input DNA, the MPS platform also offers an increased resolution of the STR regions. In the prevailing capillary electrophoresis (CE) technology, only the length of the variable strands are reported. However, for MPS the base composition for the individual sequences (also called reads) are reported. For evidential weight calculations, estimates of each compositional variant of an allele (isoalleles) needs to be estimated. Some isoalleles may not be present in the used reference database, but they may still exist in the population. We present a statistical model for estimating isoallele frequencies based on MPS data from STR markers included in the Illumina ForenSeq multiplex. For each STR marker the compositional structure is identified and their abundance observed. Based on a single step mutation model, we link the isoalleles across the varying allele lengths to estimate underlying parameters used to predict the isoallelic frequencies, both those observed in the reference database and unobserved isoalleles.

594 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P513 - KINSHIP STATISTICS: MASSIVE PARALLEL SEQUENCING vs CAPILLARY ELECTROPHORESIS Isabel Navarro-Vera1, Sara Ciria-Abad1, Sandra Esteban-Navarro1, Alba Hernandez-Maza1

1 CITOGEN, FORENSIC GENETICS, Zaragoza, Spain

The use of massive parallel sequencing (MPS) is being incorporated quickly into forensic genetics. Although it represents a much more complex and expensive technology than capillary electrophoresis (CE), its advantages are increasingly evident. The aim of this study was to compare kinship probability (W) and likelihood ratio (LR) values in paternity, maternity and specially in complex cases of indirect kinships using both MPS and CE technology. Here we present different cases (n=40) including diverse kinships: paternity, maternity, siblings, half-siblings, uncle-nephew and grandparent-grandchild. All cases were tested using AmpFl- STR® Identifiler Plus® and AmpFlSTR® NGM SElect™ PCR Amplification Kit (ThermoFischer), sequenced by CE, and ForenSeq DNA Signature Prep Kit (Verogen) sequenced by MPS. Statistic analysis were performed using the software Familias3, taking only the autosomal STRs into account and using global population allelle frequencies. Our results show that W and LR values raise dramatically not only in paternity and maternity tests, but also in complex kinship cases when using MPS. MPS allowed us to solve cases that remained unsolved with the use of the classical CE assays. Thanks to MPS we could achieve statistically conclusive results in complex kinships that remained unsolved by CE. Over 80 % of the cases studied achieved a statistically singificant raise of the W and LR values (P<0,05).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 595 P514 - LUMPING OF ALLELES IN MUTATION MODELS VASTLY IMPROVES COMPUTATIONAL EFFICIENCY Daniel Kling1, Andreas Tillmar2, Thore Egeland3

1 Oslo University Hospital, Department of Forensic Sciences, Oslo, Norway 2 National Board of Forensic Medicine and Forensic Toxicology, Department of Clinical Medicine- Linköping University, Linköping, Sweden 3 IKBM, Norwegian University of Life Sciences, Ås, Norway

Software to perform likelihood calculations in forensic genetics commonly implement various models for mutations, in particular for STR markers. A sound model from a biological perspective uses the fact that STR alleles tend to mutate through the addition or deletion of distinct repeat units,the so called stepwise model (SMM. From a computational perspective, such models are typically cumbersome and the mutation matrix can result in time-consuming calculations. Therefore, and to speed up computations, a simpler, less biologically oriented, model is often used whereby certain mathematical shortcuts can be implemented. In this study we present how alleles may be lumped and consequently the mutation matrix can be condensed. This will in the end lead to the need to traverse less genotypes in the computation algorithm. For biologically reasonable this is generally an approximation whereas for some other mutation models lumping will not affect the results. We demonstrate that by using different thresholds to perform lumping of alleles, computation speed can be vastly improved without generally having any substantial impact on the resulting likelihoods down. All the ideas will be implemented in the latest version of Familias (3.3), freely available at http://www.familias.no.

596 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P515 - MACHINE LEARNING ALGORITHMS FOR A CASE TO PREDICT Y-SNP HAPLOGROUP BASED ON Y-STR HAPLOTYPES Yiping Hou1, Mengyuan Song1, Zheng Wang1, Chenxi Zhao2

1 Sichuan University, Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Chengdu, China 2 Sichuan University, College of Computer Science, Chengdu, China

Y-chromosome single nucleotide polymorphisms (Y-SNPs) have lower mutation rate compared with Y-chromosome short tandem repeats (Y-STRs). Here we present a case about the personal identification of an unidentified cadaver using machine learning methods to determine Y-SNP haplogroup by Y-STR haplotype. Two possible haplotypes from two different male lineages were found after searching Y-STR databases. Six methods, k-Nearest Neighbor, Naive Bayesian Model, Logistic Regression, Support Vector Machine, Decision Tree, and Random Forest were used to predict the haplogroup based on Y-STR haplotype. These two haplotypes are predicted into two different haplogroups, O2a2b1a2a1 and O2a2b1a2a1a3. The predicted results were further verified by Y-SNP genotyping. It indicated that the mismatch of the two haplotypes may not originate from mutation, but due to different lineages. In this case, machine learning algorithms, especially Support Vector Machine and Random Forest showed the potential of discriminating different lineages.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 597 P516 - MASSIVELY PARALLEL SEQUENCING FOR KINSHIP ANALYSIS: HOW IMPORTANT ARE GENETIC LINKAGE CORRECTIONS? Lucinda Davenport1, David Ballard1, Laurence Devesse1, Denise Syndercombe Court1

1 King’s College London, King’s Forensics, London, United Kingdom

Traditionally, when choosing forensically relevant genetic loci targeted by capillary electrophoresis-based methods, the location of the markers relative to one another had to be considered in order to minimise the effect of genetic linkage. This allows the genotypes to be treated independently when carrying out likelihood calculations. Although these methods are largely successful at resolving paternity cases, the information they provide can often be insufficient for the analysis of more distant or complex kinship cases. Driven by this need for a greater discriminatory power, massively parallel sequencing (MPS) has been presented as an alternative to electrophoretic methods. MPS enables the simultaneous analysis of a much larger number and increased variety of markers, meaning that both STRs and SNPs can be targeted in the same reaction. MPS also provides the ability to distinguish STR alleles based on sequence rather than size, enhancing the discrimination power of most forensic STR loci. The use of MPS can be particularly beneficial when analysing complex relationship cases, where the increase in discrimination can help to clarify the specific way in which two or more individuals are related. However, when targeting multiple loci on the same chromosome, the effect of genetic linkage on the likelihood calculation must be considered. Data presented here will demonstrate that accounting for linkage can make a substantial difference to the likelihood ratio achieved, especially at the individual case level. Therefore indicating that linkage corrections should be adopted by kinship testing laboratories when increasing the number of analysed markers.

598 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P517 - MOVING FROM A CONSENSUS APPROACH TO PROBABILISTIC GENOTYPING: IMPACT ON THE VALUE OF DNA PROFILE COMPARISONS Christian Gehrig1, Basset Patrick1, Grosjean Frédéric1, Hicks-Champod Tacha1, Samie Lydie1, Castella Vincent1

1 University Center of Legal Medicine, Lausanne, Geneva, Forensic Genetics, Lausanne, Switzerland

Biological traces from casework often contain DNA from several contributors. Furthermore, resulting DNA profiles can be subject to stochastic effects, such as peak imbalance, and at its extreme allelic drop-out. To cope with these effects, our laboratory used to rely on a consensus approach based on two to four replicate analyses. When the data from a locus was not reproducible, its allelic content was not scored and only used for discrimination. This obviously represents a loss of information. In addition, in order to filter noise on the EPG in the presence of relatively large DNA quantities, but without loosing the signal from DNA if present in small amounts, an analytical threshold of 75 RFU with a general cut-off filter of 5 % was used. This simplified the DNA profiles but came with a cost. Until the end of 2018, for the comparison of DNA profiles, we only considered the presence/ absence of the scored alleles. The results of the comparisons were classified as: “Included”, “Not excluded”, “Excluded” or “Inconclusive” depending on allele sharing. Likelihood ratios (LR) were assigned using a binary approach (but ignoring stochastic effects) only when the DNA profile of the person of interest was “included” within the DNA mixture profile. Beginning January 2019, we implemented a continuous probabilistic model using allelic heights, including stutters, for each of the 16 typed locus. This also led to the discontinuation of the use of the 5 % cut-off filter. In this study, we show the results of more than 200 comparisons interpreted with our past and current methods. An increase of the cases for which a LR is assigned was observed. The pros and cons of the two approaches are discussed, as well as future developments.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 599 P518 - MOVING FROM A CONSENSUS APPROACH TO PROBABILISTIC GENOTYPING: INTEGRATION IN A LABORATORY WORKFLOW Frederic Grosjean1, Patrick Basset1, Christian Gehrig1, Hicks-Champod Tacha1, Samie-Foucart Lydie1, Vincent Castella1

1 University Center of Legal Medicine, Lausanne, Geneva, Forensic Genetics, Lausanne, Switzerland

Major change in an operational laboratory, although a pre-requisite for good science, is always a challenge. The need for the best use of traces, harmonization between different experts, combined with the ISFG commission recommendations and availability of appropriate software, triggered our change from the consensus approach to probabilistic genotyping for the comparison of complex DNA profiles with persons of interest. Using probabilistic genotyping in routine comes with its load of changes at different levels, such as data processing and communication. It necessitates adjustments for the technicians, forensic geneticists, the police and the magistrates. Here, we outline the major analyses workflow changes involved in the form of flow-charts and underline the considerations needed before switching to probabilistic genotyping. We discuss the different steps, from implementation to education, that were needed on our side so that evaluation and reporting can formally be part of our accreditation domain. In addition, we highlight the requirement for changes in proficiency testing, so that this competency can be tested. As for every new method implemented in a laboratory workflow, probabilistic genotyping has its advantages and disadvantages. We discuss the issues we encountered and show that although one should not underestimate the changes involved, the move to probabilistic genotyping is essential. Practical examples as to the impact on evaluation and in particular reporting will be given.

600 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P519 - MUTATION IN Y-STRs: REPEAT MOTIF GAINS vs. LOSSES Sofia Antão Sousa1,2,3,4, António Amorim1,4,5, Leonor Gusmão2, Nádia Pinto1,4,6

1 Institute of Pathology and Molecular Immunology from University of Porto IPATIMUP, Population Genetics and Evolution, Porto, Portugal 2 State University of Rio de Janeiro UERJ, DNA Diagnostic Laboratory LDD, Rio de Janeiro, Brazil 3 Faculty of Sciences of the University of Porto FCUP, Department of Biology, Porto, Portugal 4 Institute for Innovation and Research in Health i3S, Population Genetics and Evolution, Porto, Portugal 5 Faculty of Sciences of the University of Porto, Department of Biology, Porto, Portugal 6 Center of Mathematics of the University of Porto CMUP, Department of Mathematics, Porto, Portugal

Haploid are the only genetic systems that allow always the unambiguous identification of both parental and mutated alleles. In humans, since mtDNA is devoid of STRs, Y chromosome specific region harbors the best material to study and test mutation models of this type of forensically important markers, with the extra advantage of absence of recombination. We tested the hypothesis that at each Y-STR locus longer alleles tend to mutate to shorter ones, and vice versa, analysing 104,114 allele transfers, between father-son duos, for 19 Y-STRs. The alleles of each marker were grouped in terciles considering the number of motif repeats. The same analysis was performed pooling the markers according with their repeat motif structure: 13 GATA and 6 GAAA. In the analysis per marker, significant differences between gains and losses in first and third terciles were only observed in DYS570 and DYS626. Nevertheless, it should be remarked that significant differences between gains and losses in the second tercile were found at DYS19 and DYS439. In the analysis of the pooled loci, no evidence of differences between gains and losses was found in mutations involving “medium” (2nd tercile) alleles. In contrast, significant differences were obtained in “short” alleles for both groups (more gains than losses). Furthermore, no evidence of differentiation was found for “long” alleles at GATA markers. Moreover, we have found that average mutation rates among terciles (at both GATA and GAAA groups) are significantly different between the three allelic classes: highest for long alleles, intermediate for medium and lowest for short.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 601 P520 - OUTLIER DETECTION IN FORENSIC SCIENCE USING GRAPHICAL MODELS Mads Lindskou1, Poul Svante Eriksen1, Torben Tvedebrink1

1 Aalborg University, Department of Mathematical Sciences, Aalborg, Denmark

DNA evidence is often compared to DNA profiles from a given reference database. However, it might not be the case, that a given database is well suited for inference about a particular DNA trace. Estimation of the likelihood ratio is in general simpler if the genetic markers are independent. We have previously suggested to assess the origin of the profile of interest by a likelihood ratio test (LRT) based on Fisher’s exact test on a number of independent markers. Recent advances in genetic sequencing has made it possible to sequence short segments of DNA that contains several SNPs. These are called microhaplotypes (microhaps) and have been demonstrated applicable for ancestry assessment. The short distance between SNPs wihtin a microhap implies that recombination among them rarely occurs and that the assumption of independence no longer holds. To address this problem we exploit the family of graphical models to model the interaction between SNPs within microhaps. Given such interactions we derive a LRT for determining if a given DNA profile can be assumed to originate from a given database consisting of known DNA profiles. If a profile can not be assumed to belong to a given database, e.g. a database with profiles from a given population, the profile is said to be an outlier for that particular database. We have developed a general software for testing outliers where any number of dependent (or independent) markers can be used. In addition we have implemented an efficient procedure for estimating the interaction between markers. Interaction graphs can be used as a tool for inference about associations between markers.

602 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P521 - PERFORMANCE OF EUROFORMIX DECONVOLUTION ON POWERPLEX® FUSION 6C PROFILES Francisca Elisabeth Duijs1, Jerry Hoogenboom1, Titia Sijen1, Corina Benschop1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

Deconvolving mixed autosomal short tandem repeat profiles can be useful for database searching, profile comparisons and weight of evidence evaluations. High-template DNA profiles are readily deconvolved in case of an unmistakable major and a single minor component. For profiles with multiple and/or low-template minor contributors, the major component is generally straightforwardly extracted based on thresholds for peak heights, heterozygote balance and major to minor(s) ratio. Complete deconvolution of complex mixed profiles is far more challenging and is addressed through probabilistic interpretation software that generates a probability per genotype combination. One such software is EuroForMix that displays the deconvolved genotypes in two formats: 1) as ‘Top Marginal’ results in which for each locus a probability for the most probable (top) genotype for each contributor is given accompanied by the ratio to the next (RTN) probable genotype, and 2) as ‘All Joint’ results in which per locus a probability is presented for the considered contributors together. The performance of both approaches was examined using two- and three-person PowerPlex® Fusion 6C (PPF6C) profiles with variation for mixture proportion, amount of DNA, level of drop-out, level of allele sharing and number of replicates. By comparing the deconvolution results to the true composition of the mixtures, insight in the mechanism and performance of the EuroForMix deconvolution for PPF6C profiles is obtained. We assessed how well the All Joint probabilities correspond to the percentages of correctly inferred genotypes. Also, RTN threshold values were inferred, for instance, to achieve robust inference of the major contributor’s genotype for direct storage in a DNA database.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 603 P522 - RELATION BETWEEN STUTTER RATIO AND THE DNA SEQUENCE: FURTHER IMPROVEMENT OF EXPLAINING DISTRIBUTION OF STUTTER RATIO AT COMPLEX REPEAT LOCI Shota Inokuchi1, Koji Fujii2, Hiroaki Nakanishi1, Aya Takada3, Kazuyuki Saito1, Natsuko Mizuno2

1 Graduate School of Medicine- Juntendo University, Department of Forensic Medicine, Tokyo, Japan 2 National Research Institute of Police Science, First Department of Forensic Science- Fourth Biology Section, Chiba, Japan 3 Saitama Medical University, Department of Forensic Medicine, Saitama, Japan

The distribution of minus stutter ratio (SR) is generally explained and predicted by a positive linear correlation with Allele Repeat Number (ARN) or the Longest Uninterrupted Strength (LUS) in the repeat region of the allele. However, explaining and predicting SR with positive linear correlation is insufficient for complex repeat structure loci. To interpret capillary electrophoresis (CE) based-STR typing results using GlobalFiler Kit more robustly, we investigated the relation between SR and the DNA sequences. Furthermore, the model to predict SR at complex repeat loci from ARN as a predictor was developed. The DNA samples extracted from blood or buccal swab of one hundred eight Japanese-volunteers. The DNA sequences were analyzed by massively parallel sequencing technologies using Miseq FGx. The SR of CE-based STR typing was calculated using GlobalFiler Kit on 3500xL Genetic Analyzer. Most of the complex repeat loci (D3S1358, D8S1179, D21S11, D2S441, D13S317, D1S1656, and D2S1338) were able to classify the alleles into two or more groups where intercept of linear regression are significant differences among the group. Multiple logistic regression using ARN as a predictor was performed for estimating the group at an allele. The developed model combined with multiple linear regressions and multiple logistic regressions improved to explain and predict the distribution of SR at complex repeat locus.

604 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P523 - RELMIX: AN OPEN SOURCE SOFTWARE FOR DNA MIXTURES WITH RELATED CONTRIBUTORS Guro Dorum1, Elias Hernandis2, Thore Egeland3

1 University of Zurich, Zurich Institute of Forensic Medicine, Zurich, Switzerland 2 Universidad Autónoma de Madrid, Escuela Politécnica Superior, Madrid, Spain 3 Norwegian University of Life Sciences, Faculty of Chemistry, Biotechnology and Food Science, Aas, Norway

In both criminal cases and relationship inference there is an increasing demand for analysis of DNA mixtures where relatives are involved. The goal might be to identify the contributors to a mixture where the donors may or may not be related, or to determine the relationship between individuals based on a mixture. An example of the latter is a prenatal paternity case where the profile of the fetus is only available as a mixture with the profile of the mother. RelMix is an open source software for analysing DNA mixtures involving relatives, available as a graphical user interface in R. Relationships are represented by pedigrees and can include kinship between a number of individuals. The kinship calculations are based on the R version of Familias and allows for mutations, silent alleles and population substructure, while the mixture implementation accounts for stochastic phenomena such as dropout and drop-in. We explain the model behind RelMix and give an overview of the new features (including improved checking of input) in the latest version.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 605 P524 - THE NUMBER OF CONTRIBUTORS IS A NUISANCE (PARAMETER) FOR DNA MIXTURE EVIDENCE EVALUATION Amke Caliebe1, Klaas Slooten2

1 Kiel University, Institute of Medical Statistics, Kiel, Germany 2 VU University Amsterdam, Netherlands Forensic Institute NFI, Amsterdam, Netherlands

In the analysis of mixed DNA samples the number of contributors is typically unknown. Recently, a debate has arisen around the number of contributors postulated in hypotheses for the purpose of weight of evidence calculations on DNA mixture profiles. Should it be the same for prosecution and defence hypotheses in likelihood ratio calculations? To clarify this issue, we take the general approach of considering the number of contributors as a nuisance parameter. The form of the overall likelihood ratio depends critically on the prior distributions of the number of contributors and whether they differ between the two hypotheses. Examples are given for both scenarios where we have either independence or strong dependence between the prior distributions of the number of contributors and the hypotheses. Forthermore, we will apply the results of our analyses to point out difficulties and pitfalls in the calculation and reporting of the LR for an unknown number of contributors. The results have been published in: K. Slooten, A. Caliebe (2018) Contributors are a nuisance (parameter) for DNA mixture evidence evaluation. Forensic Science International: Genetics, Vol. 37, p 116–125.

606 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P525 - THE ROLE OF DNA CONCENTRATIONS IN FORENSIC CASEWORK RESULTS- REGRESSION MODELS APPLICATION Cláudia Vieira Da Silva1,2, António Amorim1,3, Heloísa Afonso Costa1,4, Maria João Porto1, Francisco Corte Real5,6, Marilia Antunes2,3

1 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Serviço de Genética e Biologia Forenses, Lisboa, Portugal 2 Universidade de Lisboa, Centro de Estatística e Aplicações, Lisboa, Portugal 3 Universidade de Lisboa, Faculdade de Ciências, Lisboa, Portugal 4 Universidade da Beira Interior, Faculdade de Ciências da Saúde, Covilhã, Portugal 5 Instituto Nacional de Medicina Legal e Ciências Forenses- I.P., Instituto Nacional de Medicina Legal e Ciências Forenses, Lisboa, Portugal 6 Universidade de Coimbra, Departamento de Medicina, Coimbra, Portugal

In forensic DNA typing, short tandem repeats (STRs) are the most frequently genotyped markers in order to distinguish between individuals and to relate them to a crime or to exonerate the innocent. In recent years, new controversies have arisen with the advent of more sensitive techniques, allowing profiles to be recovered from minimum amounts of DNA, hence, bringing challenges to weight of evidence evaluation for forensic DNA profiles obtained from low template DNA samples. Introduction of interpretation models, or even new weight of evidence software should be accompanied by a measure of uncertainty that is part of any biological analysis. Specially, due to stochastic effects, the reliability of the obtained profiles might differ between machinery, workflow and also PCR settings in use in different laboratories. In this work we try to understand the relation between Peak Area, DNA concentration and also size marker, using adequate regression models. Buccal swabs from 180 individuals, with unknown identity, were selected for this study. DNA was extracted with prep-n-go™ buffer and quantified using Quantifiler® Trio DNA Quantification kit in a 7500 Real-Time PCR System (Ap- plied Biosystems). STR amplification was performed with Powerplex Fusion 6C amplification kit (Promega). Amplified PCR products were separated and detected in an Applied Biosystems® 3500 Genetic Analyzer using manufacturer’s conditions. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Statistical analysis was performed with R Studio. Our results allow having an important overview of the relation between DNA concentrations, peak area, and size of the studied genetic markers.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 607 P526 - TRISOMY 21 IN PATERNITY TESTING AND FORENSIC CASEWORK Kristina Schulze Johann1, Rolf Fimmers2, Heidi Pfeiffer1, Marielle Vennemann1

1 University Münster, Institute of legal medicine, Münster, Germany 2 University Bonn, Institute of Medical Biometry- Informatics and Epidemiology, Bonn, Germany

Biostatistical assessment of samples comprising trisomy in the STR-markers located on chromosome 21 (D21-marker) could pose a challenge on common statistical methods in paternity testing and forensic casework. There is no concrete guideline how to handle these findings. Although usually there is enough information given by the STR-markers left, a trisomic finding could be important evidence in some paternity testing and crime cases and therefore might be very useful. Different case scenarios are presented and possible biostatistical evaluations are demonstrated and discussed.

608 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P527 - UNDERESTIMATION AND MISCLASSIFICATION OF MUTATIONS AT X CHROMOSOME STRs DEPEND ON POPULATION ALLELIC PROFILE Sofia Antão Sousa1,2,3,4, Eduardo Conde-Sousa5,6, Leonor Gusmão4, António Amorim1,2,3, Nádia Pinto1,2,7

1 Institute of Pathology and Molecular Immunology from University of Porto IPATIMUP, Population Genetics and Evolution, Porto, Portugal 2 Institute for Innovation and Research in Health i3S, Population Genetics and Evolution, Porto, Portugal 3 Faculty of Sciences of the University of Porto FCUP, Department of Biology, Porto, Portugal 4 State University of Rio de Janeiro UERJ, DNA Diagnostic Laboratory LDD, Rio de Janeiro, Brazil 5 Institute of Biomedical Engineering INEB, Bioimaging, Porto, Portugal 6 Institute for Innovation and Research in Health i3S, Bioimaging, Porto, Portugal 7 Center of Mathematics of the University of Porto CMUP, Department of Mathematics, Porto, Portugal

Estimation of mutation rates in X chromosome STRs is usually performed through the detection of Mendelian incompatibilities in one-generation pedigrees. However, it is known that this approach, for maximum parsimony sake, neglects the possibility of occurrence of ‘hidden’ and double (paternal and maternal) mutations as well as multistep mutations. This leads to an underestimation of mutation rates and to an overestimation of the single step over two-step changes. We have analyzed these problems using data for 8 X-chromosomal STRs, considering simulated duos and trios and real population frequency data, from Norway. For each marker, 1 000 000 simulations were computed and one and two-step mutations were considered in all possible genotypic configurations. For DXS7132, for example, when one-step mutations were simulated in trios, 13.4 % of the genotypic configurations resulted in Mendelian compatibilities. This proportion raised to 63.1 % for father-daughter, 21.0 % for mother-son, and 76.7 % for mother-daughter duos. Similarly, when two-step mutations were simulated in trios, 6.8 % of the cases resulted in Mendelian compatibilities and 17.85 % could be erroneously interpreted as one-step. For duos, these probabilities increased, respectively, to 62.9 % and 21.1 % in father-daughter; 11.1 % and 24.8 % in mother-son; 75.0 % and 19.7 % in mother-daughter. Our results not only demonstrate that current Mendelian incompatibility, maximum parsimony method underestimates both overall and multi-step mutation rates, but also show that it neglects the effect of population allele frequencies and distribution on these parameters, demonstrating that mutation detection is a function of allelic diversity at the locus in the population under study.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 609 P528 - VALIDATING THE OUTPUT OF DNA INTERPRETATION SOFTWARE USING THE CONCEPT OF CALIBRATION Jo-Anne Bright1, Mary Jones Dukes2, Simone Pugh3, Ian Evett4, John Buckleton1,5, Catherine Mcgovern1

1 ESR, Forensic Group, Auckland, New Zealand 2 QIAGEN, Human ID and Forensics, Germantown, USA 3 California DOJ, Forensic, Palo Cedro, USA 4 Principal Forensic Services, Forensic, Bromley, United Kingdom 5 University of Auckland, Statistics, Auckland, New Zealand

There is much interest in evaluating likelihood ratio (LR) performance, and hence the validity of LR assigning software. Whether performance in general, or in relation to a specific DNA profile, how do we determine that the method used is a reliable assessor of evidential weight? Exam- ining the distribution of LRs in large ground truth known trails has offered insights, however it does have limitations. Here we explore the concept of LR calibration as described in Ramos and Gonzalez-Rodriguez (2013) as a reliability assessment tool. Calibration is described as a property of a set of LRs, and its relationship to a desired set of characteristics. We interrogated a dataset of 70 QIAGEN Investigator® 24plex mixed DNA profiles. Firstly LRs were

calculated for each profile to the Hp true and 1000 Hd true donors using the software STRmix™, and performance examined via the plotting method of Taylor (2014). Secondly, LRs were converted to posterior probabilities to allow application of the calibration method. The calibration plot generated had a poorly informed mid-zone due to the high DNA profile discriminatory power. This was partially addressed by greatly increasing the false donor tests to 107 per pro-

file, and for data handling reasons focussing the analysis on a Hp true log(LR) range 5–8. We

subjectively score the calibration plot comparison of the observed frequency of Hp true and the assigned posterior probabilities as ‘good’, with all observed counts within the 0.95 bounds. This provides strong evidence for the validity of the LR assignment software with this dataset. We conclude that calibration presents a useful way forward to assess and visualize the performance of LR assigning methodologies. It is a helpful tool to complement existing assessment techniques.

610 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P529 - VALIDATION OF MASTR™ SOFTWARE: EXTENSIVE STUDY OF FULLY-CONTINUOUS PROBABILISTIC MIXTURE ANALYSIS USING POWERPLEX® FUSION 2 – 5 CONTRIBUTOR MIXTURES Teresa Snyder-Leiby1, Michael Adamowicz2, Jennifer Clarke3, Ning Wan4, Sarah Copeland1, Daniel Erb5, John McGuigan5, Chris Prosser6, James Todd6, Taylor Rambo7, Harry Makam7

1 SoftGenetics, Forensic Data Analysis, State College, USA 2 University of Nebraska–Lincoln, Director- Forensic Science Program, Lincoln, USA 3 University of Nebraska - Lincoln, Professor- Department of Statistics- Food Science and Technology, Lincoln, USA 4 SoftGenetics, Biology and Programming, State College, USA 5 SoftGenetics, Bioinformatics, State College, USA 6 SoftGenetics, Programming, State College, USA 7 University of Nebraska - Lincoln, Forensic Science, Lincoln, USA

Fully continuous probabilistic genotyping utilizes more information from the evidentiary profile; such as peak heights, stutter percentages, mixture ratio, probability of drop-in/out, than historical approaches to provide weighted genotypes. The weighted genotypes are used to calculate a likelihood ratio that a profile is included in the mixture or not. Mixture profiles are compared to hypothetical profiles created using these parameters to calculate the probability of a given individual being a contributor to the mixture. Since direct integration over a large number of interrelated parameters is not feasible, Marcov Chain Monte Carlo (MCMC) is a widely used sampling method. This validation study of MaSTR software provides the forensic community with a report of the capabilities and reliability of this tool to assist the forensic analyst in applying their expertise to evaluate mixture profiles. The validation study was designed in accordance with SWGDAM and OSAC guidelines. Puri- fied, de-identified DNA from 40 donors was obtained from Nebraska Biobank, quantified using Quantifiler® Human DNA Quantification kit, amplified with PowerPlex®Fusion, analyzed on an ABI PRISM® 3130 Genetic Analyzer, and genotyped using GeneMarker®HID software. Results of replicates and dilutions were imported into MaSTR software to calculate the variance coefficient and stutter ratios required for the protocol data set. The protocol data set provided the software with context of expected peak height variation during the evaluation of mixtures. Two, three, four, and five-contributor mixtures with a range of contributor ratios, shared alleles, peak height variance, and dilutions were tested. The elimination database was used to evaluate MaSTR software’s contamination detection capability.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 611 DNA PHENOTYPING, PHARMACOGENETICS, AND EPIGENETICS

P530 - A SYNTHETIC REAL-TIME PCR SENSOR FOR ESTIMATING THE RELATIVE LOSS OF TEMPLATE DNA DURING BISULFITE CONVERSION Harald Niederstätter1, Peter Zoufal1, Antonia Heidegger1, Catarina Xavier1, María de la Puente1,2, Walther Parson on behalf of the VISAGE Consortium1,3

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Forensic Science Program, The Pennsylvania State University, University Park- PA, USA

Methylation of cytosines plays a crucial role in (human) epigenetics, e.g. by modulating gene expression without changing the underlying nucleotide sequence. Methylation patterns may be inherited (“parental imprinting”) and they are subject to changes due to age and/or exposure to environmental factors. This triggered great interest in the pure and applied bio-medical sciences. In forensic epigenetics, DNA methylation profiling is mainly restricted to tissue-of-origin determination, age estimation, and molecular genetic resolution of identical twins, but additional fields of application are emerging [1]. Bisulfite conversion is a popular technique to study DNA methylation by converting unmethylated cytosines to uracil (which is typically replaced by thymine during PCR) while leaving methylated cytosines unaltered. Downstream sequencing analysis of PCR products allows for cytosine methylation profiling at single base resolution. Yet, harsh physico-chemical conditions encountered in the conversion process lead to DNA fragmentation and purification of conversion products causes further loss of template DNA. To monitor such losses, we set out to develop a relative quantitative real-time PCR assay relying on an artificial, spiked-in sensor system. We will discuss the major design considerations and show some key characteristics of the assay as determined experimentally. [1] Vidaki A, Kayser M, Genome Biol (2017) 18:238. The study received support from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 740580 within the framework of the VISAGE project and consortium.

612 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P531 - AGE PREDICTION METHOD BASED ON DNA METHYLATION FOR USE IN FORENSIC ANALYSIS Benjamin Franken1, Anke Prochnow2, Keith Elliott3, Charline Bemmann1, Andrea Janosch1, Norbert Hochstein1

1 QIAGEN GmbH, Research & Development, Hilden, Germany 2 QIAGEN GmbH, Global Product Management, Hilden, Germany 3 QIAGEN, Strategic Marketing, Manchester, United Kingdom

Age prediction has the potential to provide vital intelligence in criminal investigationsinvolv- ing non-suspect situations, and where DNA profiles do no match against databases. Previous studies have identified several loci in the genome that show a strong correlation between DNA methylation frequency of CpG sites and chronological age, but currently forensic investigators are lacking a stable and reproducible workflow for age prediction that can be easily integrated into their laboratory. In this study we present the development of a flexible and easy-to-use workflow for age prediction using novel Pyrosequencing technology. From multiple candidate loci showing linear correlation with chronological age in blood we have chosen the five most significantly correlated CpG sites for our development (ELOVL2, C1orf132, TRIM59, KLF14 and FHL2) [1]. We optimized two important steps: the amplification of the candidate loci and the analysis of DNA methylation. In order to efficiently use the limited amount of sample DNA and to reduce the hands-on-time per sample, we have established a multiplex strategy for the amplification of the five markers. For DNA methylation analysis we used the PyroMark Q48 Autoprep System. This Pyrosequencing® platform simplifies the workflow and increases the throughput by offering integrated, automatic sample preparation. Our results outlines a clear and simplified workflow that can easily be adapted for future challenges in this field, e.g. the development of novel markers for age prediction in variable tissue species. References [1] Renata Zbieć-Piekarska et al., (2015) Development of a forensically useful age prediction method based on DNA methylation analysis. FSI: Genetics 17:173–179

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 613 P532 - AN ENHANCED SNP MULTIPLEX FOR THE PREDICTION OF HUMAN PIGMENTATION BY COMBINING ESTABLISHED GENOTYPING ASSAYS Manuel Fondevila Alvarez1, Zaira García López1, Olalla Lòpez Costas2, Christopher Phillips1, María Victoria Lareu Huidobro1

1 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic genetics unit, Santiago de Compostela, Spain 2 University of Santiago de Compostela, Dept Edafología y Química Agrícola, Edafología y Química Agrícola, Santiago de Compostela, Spain

The prediction of human pigmentation traits remains the key approach to current forensic analysis of externally visible characteristics. While the SNP variants that affect the expression of a particular pigmentation phenotype in humans have been extensively studied, the predictive power of any one test varies depending on the particular phenotype that is predicted. Extreme phenotypes are consistently predicted with higher statistical values and with less error, than intermediate phenotypes. This factor can be explained by the presence of just a few SNPs whose genotype is strongly linked to an extreme phenotype, such as blue vs. brown eyes. For intermediate phenotypes (such as light brown eye colour, or hair colours within the blond-brown spectrum) the situation is more complex; with a lack of major SNPs that predict intermediate phenotypes. This phenomenon can be explained by these phenotypes having a wider range of underlying SNPs with reduced individual effect. To raise the predictive values obtained for human pigmentation phenotypes, we have merged two independently developed forensic assays that predict eye and hair colour: HIrisPlex and USC´s SHEP assays. A combined test composed of 42 SNPs from these two systems has been optimized with two parallel multiplexed PCR-SNaPshot reactions genotyping all HIrisPlex and SHEP SNPs. The primer and probe sequences have been taken from the original publications with minor modifications made to accommodate the full set of SNPs to a common electrophoretic separation. The new multiplexes genotype all the key SNPs related to eye and hair colour, and consequently maximize the predictive values obtained from forensic DNA analysis.

614 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P533 - AN EXTENDED AGE PREDICTION MODEL FROM CHILDHOOD TO THE ELDERLY Ana Freire-Aradas1, Lorena Girón-Santamaría1, Ana Mosquera Miguel1, Adrián Ambroa1, Christopher Phillips1, María de los Ángeles Casares de Cal2, Antonio Gómez‑Tato2, Jose Álvarez‑Dios2, Ewelina Pośpiech3, Anastasia Aliferi4, Denise Syndercombe Court4, Wojciech Branicki3, Ángel Carracedo1, María Victoria Lareu1

1 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic Genetics Unit, Santiago de Compostela, Spain 2 University of Santiago de Compostela, Faculty of Mathematics, Santiago de Compostela, Spain 3 Jagiellonian University, Malopolska Centre of Biotechnology, Kraków, Poland 4 King’s College London, Faculty of Life Sciences and Medicine, Department of Analytical- Environmental and Forensic Sciences, London, United Kingdom

Forensic age estimation is a DNA intelligence tool that forms an important part of Forensic DNA Phenotyping. Criminal cases with no suspects or with unsuccessful matches in searches of DNA databases; human identification analyses in mass disasters; or anthropological studies all benefit from age estimation to gain investigative leads. Several age prediction models have been developed to date based on DNA methylation. Al- though different DNA methylation technologies as well as diverse statistical methods have been proposed, most of them are based on bloodstains and mainly restricted to adult samples. In the current study, we present an extended age prediction model based on 895 evenly distributed DNA blood samples from 2 to 104 years old. DNA methylation levels were detected using Agena bioscience EpiTYPER® technology for a total of 7 CpG sites located at seven genomic regions: ELOVL2, ASPA, PDE4C, FHL2, CCDC102B, MIR29B2CHG and chr16:85395429 (GRCh38). The accuracy of the age prediction system was tested by comparing three statistical methods: quantile regression analysis versus two machine learning algorithms.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 615 P534 - APPLICATION OF THE ION AMPLISEQ™ HIRISPLEX-S METHOD FOR PREDICTING PIGMENTATION TRAITS IN DEGRADED BONE SAMPLES Magdalena Kukla-Bartoszek1, Maria Szargut2, Ewelina Pośpiech3, Grażyna Zielińska2, Marta Diepenbroek2,4, Magdalena Spólnicka5, Wojciech Branicki3, Andrzej Ossowski2

1 Malopolska Centre of Biotechnology Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Poland 2 Pomeranian Medical University in Szczecin, Department of Forensic Genetics, Szczecin, Poland 3 Jagiellonian University, Malopolska Centre of Biotechnology, Krakow, Poland 4 Universität München, Institut für Rechtsmedizin der Universität München, Munchen, Germany 5 Central Forensic Laboratory of the Police, Biology Department, Warszawa, Poland

Identification of human remains is the important area of modern forensic genetics. In many studies of skeletal remains predictive DNA analysis can support the process of human identification by providing important investigative guidance. The field of forensic DNA phenotyping is progressing significantly and the recently introduced HIrisPlex-S method allows simultaneous prediction of eye, hair and skin colour. Due to the common degradation of skeletal samples, their analysis may be a challenge. In this study we used the Ion AmpliSeqTM HIrisPlex-S panel and Ion Torrent technology to analyze bone samples that exhibit different levels of DNA degradation. In total, 63 bone samples at post-mortem intervals from 1 to above 70 years were genotyped and eye, hair and skin colour predictions were done using HIrisPlex-S (HPS) models. Complete HIrisPlex-S profiles were generated from as little as 50 pg of template DNA. In total, the whole set of 41 predictors was generated in 34 samples (54.0 %) when coverage threshold ³200 reads/SNP was applied. Twenty-four samples produced partial profiles and clearly showed underperformance of 3 HIrisPlex-S SNPs: rs1545397 and rs1470608 in OCA2 and rs10756819 in BNC2. Of these 24 samples, eye colour prediction was possible for 20 cases (83.3 %) and hair colour prediction for 18 cases (75.0 %) with no missing data for SNPs included in the corresponding models. For none of these samples full profile for skin colour SNPs was obtained and therefore no prediction was possible. Results obtained using NGS technology were additionally compared with the outcome of STR analyses. The study shows that DNA degradation and the resulting loss of data are the most serious challenge for DNA phenotyping of skeletal remains.

616 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P535 - BIOGEOGRAPHICAL ANCESTRY PREDICTION ACCURACY – A SWEDISH PERSPECTIVE Adam Staadig1,2, Klara Junker3, Maja Sidstedt3,4, Johannes Hedman3,4, Andreas Tillmar1,2

1 Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden 2 Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden 3 National Forensic Centre, Swedish Police Authority, Linköping, Sweden 4 Applied Microbiology- Department of Chemistry, Lund University, Lund, Sweden

Novel methods have been developed to expand the use of forensic DNA, such as SNP-based phenotyping. This includes prediction of the biogeographical ancestry of the unknown donor, which can supply the police with useful investigative leads. Before introducing such methods in routine casework, it is however important to thoroughly evaluate their performance. In this study, we examined the biogeographical ancestry prediction accuracy for 78 individuals from a Swedish population data set and for another 111 individuals residing in Sweden with various population origins. All individuals were typed for the 56 ancestry informative SNPs included in the ForenSeq DNA Signature Prep kit (Verogen) using massively parallel sequencing. The predictions of biogeographical ancestry were performed with four established prediction models; multinomial logistic regression (MLR), naïve Bayes, principal component analysis (PCA) and STRUCTURE. The predictions were based on published reference data which comprised of a total of 2,651 individuals from 28 different populations from six different continents. In general, all prediction methods performed similarly with average AUC above 0.8 and median prediction probabilities in the range of 0.99. Furthermore, although predictions with STRUCTURE overall were slightly less informative for non-admixed individuals, it performed better for admixed individuals compared with both MLR and naïve Bayes. We conclude that it is better to use the combined information from multiple predictors rather than to rely on one single ancestry prediction method.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 617 P536 - BMI PREDICTION THROUGH DETECTION OF DNA METHYLATION Lucie Kotková1, Rastislav Slavkovský1, Veronika Holinková1, Barbora Blumová1, Helena Štefanová1, Jana Stránská1, Jiří Drábek1

1 Palacky University Olomouc, Institute of Molecular and Translational Medicine, Olomouc, Czech Republic

DNA methylation is the most widely used epigenetic modification in a forensic setting. Its changes may arise as a result of lifestyle and environmental factors. Thus, epigenetics can contribute not only to the estimation of the tissue origin in the biological sample but also help predict various phenotypical characteristics of the trace donor. The most important phenotypic characteristics tested by now is age, but markers for other characteristics like time of trace deposition, donor smoking, alcohol consumption, or diet are being searched for as well. These predictions can lead the investigation by narrowing down the population from which sample donor recruits. In medical and obesity research, a correlation between methylation of specific gene loci and body mass index (BMI, a measure for indication nutritional status in adults) was found. The methylation status of these markers could predict body structure of an unknown sample donor. We decided to verify a correlation between BMI and methylation status in three previously validated CpGs in HIF3A gene and other previously published candidate markers using a group of healthy Czech blood donors (n>20) with BMI under 21 kg/m2 (n>10) and over 36,5 kg/m2 (n>10). For methylation assessment, we choose next-generation amplicon bisulfite sequencing over the pyrosequencing method, mostly because of lower sample requirements (0.5 ng DNA for NGS vs 10 ng DNA using PyroMark method) which are in a lot of cases the bottleneck for the usage of the epigenetic methods in the forensic setting. Here we present our preliminary data. Acknowledgements: Supported by the European Regional Development Fund – Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868), CZ.02.1.01/0.0/0.0/16_013/0001674, IGA_ LF_2019_003, and NPU LO1304.

618 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P537 - BUILDING THE BRIDGE BETWEEN SCIENCE AND INVESTIGATION Runa Daniel1, Dennis McNevin2, Alison Sears3, Jennifer Raymond4

1 Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Victoria, Australia 2 Centre for Forensic Science-School of Mathematical and Physical Sciences MaPS- Faculty of Science, University of Technology Science, New South Wales, Australia 3 Forensic and Analytical Science Service Laboratory, NSW Health Pathology, New South Wales, Australia 4 Forensic and Technical Services Command, NSW Police Force, New South Wales, Australia

In the era of intelligence-led policing, significant police and forensic resources are directed towards expanding and enhancing intelligence capabilities. DNA Intelligence, also known as forensic DNA phenotyping, enables the prediction of biogeographical ancestry and externally visible characteristics of the donor of biological evidence. The increased accuracy in DNA In- telligence results from the application of massively parallel sequencing (MPS) which enables the analysis of hundreds of DNA markers simultaneously. In the absence of an STR profile match, DNA Intelligence assists in generating or re-prioritising a suspect pool. Its application includes criminal investigations and coronial matters. Reliable application of DNA Intelligence requires interpretation and communication of the scientific data to an investigator. The communication should provide a clear indication of the intelligence and its reliability. Engagement with detectives and Intel analysts through the application of DNA Intelligence in casework, educational seminars and feedback surveys highlighted the need for a common language to bridge the gap between science and investigation. The requirement for expert scientific skills in interpretation of the data was also identified. The international Intelligence community uses Words of Estimative Probability (WEP) in Intelli- gence analytical reports to provide clear estimates to establish strategies or make decisions. We investigated a novel approach to reporting DNA Intelligence from the MPS-based Precision ID Ancestry panel (Thermo Fisher Scientific) using WEP. Ancestry data estimates were aligned to WEP scales. We discuss the development of interpretation and reporting guidelines to assist in reliable application of DNA Intelligence in investigations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 619 P538 - DEVELOPMENT AND VALIDATION OF THE VISAGE TOOLS TO ESTIMATE AGE BY MASSIVELY PARALLEL SEQUENCING Antonia Heidegger1, Catarina Xavier1, Harald Niederstätter1, María de la Puente1,2, Ewelina Pośpiech3, Aleksandra Pisarek3, Manfred Kayser4, Wojciech Branicki3,5, Walther Parson1,6

1 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 2 Forensic Genetics Unit - Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland 4 Erasmus MC University Medical Center Rotterdam, Department of Genetic Identification, Rotterdam, Netherlands 5 Central Forensic Laboratory of the Police, Central Forensic Laboratory of the Police, Warsaw, Poland 6 The Pennsylvania State University, Forensic Science Program, Pennsylvania, USA

The prediction of externally visible characteristics of an unknown sample donor can provide essential investigative leads if no reference material for conventional genetic identification is available. To make such predictions feasible in forensic routine laboratories, the VISible Attributes through GEnomics (VISAGE) Consortium aims to develop and forensically validate new tools to broaden the use of DNA intelligence towards the construction of composite sketches from biological traces. This includes not only the determination of appearance and ancestry markers, but also the estimation of the sample donor’s age. Information on age can help to guide police investigations and adds detail to phenotypic traits like hair loss, hair greying and skin aging features. The VISAGE age estimation tools are based on massively parallel sequencing of carefully selected DNA methylation markers. In the first phase of this project, we developed a basic tool for age estimation from blood traces. It targeted 5 CpG markers in one PCR multiplex assay for bisulfite converted DNA, followed by sequencing with the MiSeq FGx. Here we present results from this basic tool that has undergone forensic validation including inter-laboratory testing. The second phase of this project is currently ongoing and dedicated to the development of an enhanced tool comprising an enlarged number of markers to improve prediction accuracies and enable age estimation from additional forensically relevant biological materials. The study received support from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 740580 within the framework of the VISAGE Project and Consortium.

620 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P539 - DEVELOPMENT OF A DNA METHYLATION BASED AGE ESTIMATION MODEL INCLUDING 4 CPG SITES Kristina Schwender1,2, Marianne Schuerenkamp1, Marielle Vennemann1, Qiagen GmbH3, Heidi Pfeiffer1

1 Westfaelische Wilhelms-University of Muenster, Institute of Legal Medicine, Muenster, Germany 2 Ludwig-Maximilians-University of Munich, Institute of Legal Medicine, Munich, Germany 3 Qiagen, GmbH, Hilden, Germany

The analysis of DNA methylation levels of specific CpG sites seems to be one of the most promising techniques to estimate an individual’s biological age. Many studies were published presenting age estimation models based on DNA methylation patterns from blood samples, only a few studies used saliva or buccal swab samples. In this study 88 CpG sites in 8 different genes were analyzed in buccal swab samples from 95 individuals with ages ranging from 20 to 69 years by pyrosequencing (PyroMark Q48 Autoprep, Qiagen). Stepwise linear regression analysis was performed resulting in an age estimation model including 4 CpG sites located in the genes PDE4C, EDARADD, SST and KLF14. Additionally, a minisequencing multiplex reaction (SNaPshot Multi- plex Kit, Thermo Fisher Scientific) comprising the 4 CpG sites mentioned above was developed, providing a cost-effective method for DNA methylation analyses in forensic laboratories. Model performance, results and correlation of the two methods will be presented and discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 621 P540 - DEVELOPMENT OF A FORENSIC AGE PREDICTOR FOR SALIVA/BUCCAL SWABS BASED ON DNA METHYLATION Adrián Ambroa1, Lorena Girón-Santamaría1, Ana Mosquera Miguel1, Christopher Phillips1, María de los Ángeles Casares de Cal2, Antonio Gómez-Tato2, Jose Álvarez-Dios2, María Victoria Lareu1, Ana Freire-Aradas1

Age estimation can provide key information in criminal, legal and anthropological investigations. Sometimes the donor of a biological stain cannot be identified; so estimation of the individual age can contribute to a reduction in the number of suspects in an investigation. Currently, most forensic age prediction models developed are based on blood samples, but this is not the most common body fluid encountered in forensic analyses. Therefore, the development and optimization of individual age prediction models for additional tissues present in crime scenes such as saliva is an essential next step. Currently, there is a range of techniques that allow the detection of DNA methylation. SNaP- shotTM is a technology that can be used for such analyses by including an initial pre-treatment of DNA with sodium bisulfite. The main advantage of this technique is its ability to analyze multiplexed DNA fragments, providing rapid detection of several CpG positions in a single analysis. In addition, SNaPshot tests require low amounts of DNA and can be performed with short PCR amplicons, which provides a significant advantage for analyzing degraded samples. In our study, the selection of candidate age-correlated CpG sites was performed using either public data from Illumina HumanMethylation 450 BeadChip array-based studies or previous literature. A single-tube SNaPshot reaction was optimized, and DNA methylation levels were tested for saliva and buccal swab samples for volunteers between 23 and 86 years old. A preliminary age prediction model has been developed for the detected methylation patterns.

622 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P541 - DEVELOPMENT OF A FORENSIC DNA PHENOTYPING PANEL USING MASSIVE PARALLEL SEQUENCING Chiara Turchi1, Valerio Onofri1, Filomena Melchionda1, Adriano Tagliabracci1

1 Polytechnic University of Marche, Department of Biomedical Sciences and Public Health, Ancona, Italy

Forensic DNA Phenotyping (FPD) refers to the prediction of human appearance traits directly from biological materials and it has become, in the last years, an important field in forensic genetics. It allows to infer the unknown perpetrator’s externally visible characteristics (EVCs) directly from DNA traces left behind at the crime scene and it is also useful for unknown deceased missing person identifications, cold cases and to evaluate phenotypic characteristics of skeletal remains. The HIrisPlex-S system, targeting a total of 41 SNPs, allows the simultaneous eye, hair and skin colour prediction from DNA and it is available in the public website (https://hirisplex.erasmusmc. nl/). In the present study, we developed a massive parallel sequencing (MPS) multiplex assay in order to genotype all the 41 SNPs, included in the HIrisPlex-S system, in degraded forensic casework samples. The MPS panel was designed through the Ion AmpliSeq Designer web tool. PCR amplicons sizes of target regions were kept below 180 bp, allowing application to degraded DNA typically found in forensic samples. MPS were performed by Ion PGM and Ion Gene Studio S5 Sys- tem. Different types of forensic casework samples such as blood, saliva stains, bone remains and touch DNA samples were tested in order to evaluate the concordance testing, the robustness and forensic suitability of this 41-plex assay.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 623 P542 - DEVELOPMENT OF ANCESTRY INFORMATIVE MARKER SETS FOR THE VISAGE BASIC AND ENHANCED MPS TOOLS Christopher Phillips1, Ana Freire-Aradas2, Ana Mosquera Miguel3, María de la Puente2, María Victoria Lareu2, Ángel Carracedo2, Wojciech Branicki4, Michael Nothnagel5, Walther Parson6, Manfred Kayser7, The VISAGE Consortium7

1 Institute of Forensic Sciences- University of Santiago de Compostela, Forensic Genetics Unit, Santiago de Compostela, Spain 2 Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 3 Forensic Genetics Unit- Institute of Forensic Sciences, Univeristy of Santiago de Compostela, Santiago de Compostela, Spain 4 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland 5 Cologne Center for Genomics CCG, University of Cologne, Cologne, Germany 6 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 7 Department of Genetic Identification, Erasmus MC University Medical Center, Rotterdam, Netherlands

The VISAGE Consortium has developed an all-in-one analysis system for forensic DNA phenotyping (FDP) using massively parallel sequencing (MPS). This system delivers the core ‘triple-A’ FDP data about unidentified crime-scene DNA donors: Appearance, Ancestry, Age - then applies the genetic information in a privacy-by-design software toolbox to predict these characteristics. The particularities of methylated DNA preparation demand a separate MPS pipeline and assay for age estimation compared with one that combines ancestry markers (AIMs) and SNPs for common externally visible characteristics (EVCs) to reconstruct a suspect’s likely appearance. Here we detail the steps taken to develop AIM sets for the VISAGE Basic Tool (BT) and Enhanced Tool (ET), and their constituent locus characteristics. The VISAGE BT comprises 99 binary AIM-SNPs, each with a population indicative allele for one of five main continental population groups and South Asian and Middle East groups; plus 15 tri-allelic SNPs with two indicative alleles. The 114 AIM-SNPs were successfully combined with 38 EVC-SNPs to create a 152-plex assay, whose forensic performance is reported in another presentation. The VISAGE ET was developed to extensively enhance AIM number, population differentiation power and locus type, as well as major extension of EVC traits and their predictive loci. Continental population AIM-SNPs were halved from 68 to 34 to release multiplex space for other AIMs, comprising: 85 Y-SNPs informative for a global phylogenic tree; 16 X-SNPs with a 5-SNP centromeric haplotype motif for co-ancestry inference in admixed persons; 15 South Asian informative SNPs (19 in BT); 29 Middle East informative SNPs (12 in BT); 26 tri-allelic SNPs; and 21 microhaplotypes - enhancing mixed-DNA detection using ET.

624 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P543 - DEVELOPMENT OF BMI-ASSOCIATED PREDICTION MODELS FOR A KOREAN POPULATION: A COMPARISON WITH EWAS STUDY Ji Hyun Lee1, Eun Hee Lee2, Hye In Kim3, Yu Na Oh4, Jeong Min Lee1, Jae Jun Ahn3, Eun Ho Ha3, Hwan Young Lee1,5

1 Seoul National University College of Medicine, Department of Forensic Medicine, Seoul, Republic of Korea 2 Yonsei University College of Medicine, Department of Forensic Medicine, Seoul, Republic of Korea 3 Yonsei University, Department of Information Statistics, Wonju, Republic of Korea 4 Ministry of National Defense, Division of DNA Analysis- Scientific Investigation Laboratory- Criminal Investigation Command, Seoul, Republic of Korea 5 Seoul National University College of Medicine, Institute of Forensic and Anthropological Science, Seoul, Republic of Korea

When DNA profiles obtained from biological evidences at a crime scene fail to match suspects in the database, prediction of phenotyping can facilitate a traced search for an unknown suspect by limiting the search range. DNA methylation has been known to change according to the complex traits such as alcohol consumption, smoking behavior, body mass index (BMI) etc., and therefore, has been used to develop molecular predictors for a multitude of traits and disease. In the present study, for the development of a prediction model for BMI, we investigated several previously reported BMI or obesity-associated genetic and epigenetic markers including 4 CpGs (ABCG1, CPT1A, LMNA and PDE4DIP genes) and 8 SNPs (near the genes, FTO, BDNF, SEC16B, MC4R, TMEM18, GIPR/QPCTL, ADCY3/RBJ and GNPDA2) in 700 Koreans. Using data mining techniques, i.e. decision tree (entropy and gini), random forest and bagging, a total of eight models that have 31 or 32 BMI as a cut-off value were constructed with age and sex as a co- variate, and among them, a random forest model with a cut-ff value of 31 showed the highest prediction accuracy of 63 %. Additionally, a linear regression function modeled showed that the 4 CpG sites explains 11.5 % total variance in BMI with an error of 4.25. These results were very similar to those of the previous research by McCarthy et al., where a regression model constructed using the DNA methylation data at 1109 CpG sites showed an R-squared value of 0.125 in more than 5,000 people. Although the accuracy for prediction of BMI was not high, our study is meaningful in a respect that a small number of markers can achieve similar prediction accuracy to that obtained from a model constructed with EWAS analysis.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 625 P544 - DNA METHYLATION-BASED AGE PREDICTION FROM SKELETAL REMAINS Ji Eun Lee1, Sae Rom Hong2, Nam Ye Kim3, In Kwan Hwang3, Sang-Eun Jung2, Dong Sub Lee3, Yang Han Lee3, Pil Won Kang3, Hwan Young Lee1,4

1 Seoul National University College of Medicine, Department of Forensic Medicine, Seoul, Republic of Korea 2 Yonsei University College of Medicine, Department of Forensic Medecine, Seoul, Republic of Korea 3 National Forensic Service, Forensic DNA Division, Wonju, Republic of Korea 4 Seoul National University College of Medicine, Institute of Forensic and Anthropological Science, Seoul, Republic of Korea

Age prediction can help identifying skeletal remains by limiting the search range for a missing person. Age prediction methods based on odontology and archaeology have been frequently used in the forensic field, but DNA methylation is currently known to be one of the most promising age-predictive biomarkers. In the present study, we generated genome-wide DNA methylation profiles of human compact bone samples obtained from 32 deceased individuals who were identified by STR analysis. Age at death of 32 remains were assumed to be 31 to 96 years. Only 12 of 32 samples produced more than 840K quality-filtered CpG methylation values in Illu- mina’s Infinium MethylationEPIC BeadChip array analysis, and another 9 samples produced more than 720K quality-filtered CpG values. Methylation ages of the 12 samples were calculated using a recently developed epigenetic clock composed of 391 age-associated CpGs, but the other samples could not be used because of high number of missing values. In a test for age association of the CpG sites included in the new epigenetic clock using DNA methylation array data obtained from 12 bone DNA samples and 54 saliva DNA samples, several pan-tissue age-associated markers, such cg16867657, cg06639320 and cg07553761 of the ELOVL2, FHL2 and TRIM59 genes showed strong age correlation both in bone and saliva DNA samples, but their methylation levels were considerably different from each other. When considering the very low success rate observed in methylation beadchip array and different methylation levels between bone and other tissues, it would be needed to discover a new set of a few CpG sites with a high age correlation for age prediction in skeletal remains.

626 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P545 - EARLY EVALUATION OF FIVE AGE-CORRELATED DNA METHYLATION MARKERS IN AN ITALIAN POPULATION SAMPLE Fabiano Gentile1, Elisa Castoldi1, Patrizia Serventi1, Domenico Colloca1, Roberto Ciccotelli1, Alberto Marino1

1 Reparto Carabinieri Investigazioni Scientifiche di Parma, Sezione Biologia, Parma, Italy

Age prediction helps investigation to narrowing down the number of potential suspects and give more reliable predicted appearance. However, since it is not known if some of the widely used methylation markers (ELOVL2, C1orf132, TRIM59, KLF14 and FHL2) are applicable as age predictors in the Italian population, in this study we collected and analyzed a set of saliva samples from 96 Italian individuals aged 19 to 67 years. All DNA samples were quantified, bisulfite-converted, amplified with monoplex and analyzed using PyroMark Q48 Autoprep (Qiagen). The age prediction was performed according to reference1. Preliminary results shown a linear correlation between the methylation markers studied and age in the Italian samples, with a Pearson correlation coefficient (PCC) of 0.71 and the value of median absolute deviation (MAD) of 5.4. The MAD value observed with group 1 (age 20s or less) was 2.8, with group 2 (30s) was 4.5, with group 3 (40s) was 4.7 and with group 4 (50s or more) was 5.4. If we take the assumption that age prediction is correct when predicted age is in the range of max ±5 years of the actual age, the number of correct prediction decrease with increasing of the age group. Moreover, in contrast of what expected, our data set has shown a bias in the underestimating of predicted age at the increasing of chronological age (20 % group 1, 60 % group 2, 79 % group 3 and 88 % group 4). This experiment suggest to increase the number of different population sampled since DNA methylation could be affected by genetic variation, environmental and lifestyle variation. 1. Zbieć-Piekarska, R. et al.: Development of a forensically useful age prediction method based on DNA methylation analysis. FSI:Genetics, 2015;17:173–9

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 627 P546 - EFFICIENCY OF EYE COLOUR PREDICTION ALGORITHMS IN CASE OF HIGH FREQUENCY OF INTERMEDIATE COLOUR: AN ITALIAN STUDY Cecilia Salvoro1, Christian Faccinetto2, Luca Zucchelli1, Marika Porto1, Alberto Marino2, Gianluca Occhi1, Gustavo de los Campos3, Giovanni Vazza1, Fabiano Gentile2

1 University of Padova, Department of Biology, Padova, Italy 2 Reparto Carabinieri Investigazioni Scientifiche di Parma, Sezione Biologia, Parma, Italy 3 Institute for Quantitative Health Science and Engineering, Michigan State University, Departments of Epidemiology, Biostatistics and Statistics, Probability, East Lansing, Michigan, USA

The majority of models for Forensic DNA phenotyping have been developed for iris color; but, replication studies to understand their applicability on a worldwide scale are still limited for many of them. In our work 4 models for eye color prediction (IrisPlex, Ruiz, Allwood and Hart) were systematically evaluated in a sample of 296 subjects of Italian origin. Genotypes were determined by a custom NGS-based panel with all the predictive SNPs included in the 4 tested models. Overall, 60–69 % of the Italian sample could be correctly predicted with the IrisPlex, Ruiz and Allwood models, applying the recommended threshold. The IrisPlex model showed the lowest frequency of errors (17 %), but the highest number of inconclusive results (18 %). Without threshold, the correct predictions obtained were 76 % for IrisPlex, 73 % for Allwood and 65 % for Ruiz. (The Hart model had the lowest error rate (2 %), but the 87 % of predictions were restricted to the less informative categories of “not-blue” and “not-brown”.. As just observed, the majority of incorrect and undeﬁned predictions were ascribable to the intermediate category, which represented 25 % of the Italian sample. An adjustment of the IrisPlex (multinomial logistic regression) and Ruiz models (Snipper Bayesian classiﬁer) with Italian allele frequencies gave only minor improvements in predicting intermediate eye color and no remarkable overall changes in performance. This suggests an incomplete knowledge underlying the intermediate colors. Considering the impact of this phenotype in Italian samples as well as in other admixed populations, future improvements of eye color prediction methods should include a better genetic and phenotypic characterization of this category. FSI: Genetics 40 (2019) 192–200.

628 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P547 - ENHANCED CELL-TYPE INFERENCE BY QUANTITATIVE “ON/OFF” CPG METHYLATION PROFILING Christian Gausterer1, Sahar Houshdaran2, Christina Stein1, Gerda Egger3

1 Forensisches DNA Zentrallabor GmbH, Medical University of Vienna, Vienna, Austria 2 Obstetrics- Gynecology and Reproductive Sciences, University of California, San Francisco, USA 3 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria

Over the past decade “source tracking” based on tissue-specific differentially methylated regions (tDMRs) has gained attention in the forensic scientific community as a modality for identifying the cellular origin (tissue, body fluid) of purified genomic DNA. Here we evaluated a modular multistep approach that involves digestion of native (i.e. non-sodium bisulfite-treated) DNA using methylation-sensitive restriction enzymes followed by qualitative “endpoint” PCR (MS- RE-PCR) and quantitative real-time PCR (MSRE-qPCR), respectively. Various multiplex formats were developed in order to address different tasks: firstly, the “Screening-plex” (8-plex MSRE-PCR) employing five biofluid-specific markers (for the discrimination of human blood, saliva, vaginal fluid and semen) plus three control markers (PCR, digestion); secondly, the “SF-plex” (6-plex MS- RE-PCR) for the detection of semen targeting three discriminatory loci (semen-specific versus “non-semen”) plus controls; thirdly, the “BL-plex” (6-plex MSRE-PCR) for the detection of blood investigating three discriminatory loci (blood-specific versus “non-blood”) plus controls. Valida- tion of MSRE-PCRs was performed including sensitivity, specificity and mixture identification. Furthermore, hydrolysis probe-based MSRE-coupled qPCR multiplex assays were established to quantitatively examine different combinations of discriminatory loci (as indicated by results from qualitative analysis) plus controls. Thereby, cell-type deconvolution in complex samples that contain both methylated and unmethylated fractions was vastly improved. Our technology allows for the sensitive and specific identification of body fluids in trace amounts in forensic samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 629 P548 - EPIGENETIC AGE ESTIMATION IN SEMEN SAMPLES – ON THE WAY TO GOOD MARKERS AND METHODS Aleksandra Pisarek1, Ewelina Pośpiech1, Anna Papież2, Antonia Heidegger3, Catarina Xavier3, Ramya Potabattula4, Marta Sikora-Polaczek5, Aneta Macur6, Jarosław Janeczko6, Thomas Haaf4, Joanna Polańska2, Walther Parson3,7, Manfred Kayser8, Wojciech Branicki1,9, on behalf of the VISAGE Consortium

1 Jagiellonian University, Malopolska Centre of Biotechnology, Krakow, Poland 2 The Silesian University of Technology, Data Mining Division, Gliwice, Poland 3 Medical University of Innsbruck, Institute of Legal Medicine, Innsbruck, Austria 4 Julius Maximilians University, Institute of Human Genetics, Würzburg, Germany 5 Medical Centre Macierzyństwo, Medical Centre Macierzyństwo, Krakow, Poland 6 PARENS Fertility Centre, PARENS Fertility Centre, Krakow, Poland 7 The Pennsylvania State University, Forensic Science Program, Pennsylvania, USA 8 Erasmus MC University Medical Center Rotterdam, Department of Genetic Identification, Rotterdam, Netherlands 9 Central Forensic Laboratory of the Police, Department of Biology, Warsaw, Poland

Age information may be useful in a criminal investigation for intelligence purposes. It is known that epigenetic changes constitute an important component of ageing processes and in particular, DNA methylation has been demonstrated to serve as a very accurate measure of an individual’s age. Most of the studies have focused on somatic cells and although the identified CpG markers allow accurate age estimation in blood and saliva, the differences in DNA methylation pattern of sperm cells prevent their use to predict age in semen. To overcome this limitation, a novel discovery analysis was conducted using Illumina 850K EPIC Infinium MethylationEPIC BeadChip microarrays. The data were collected for 40 semen samples from men aged 24–58. Multivariable linear regression on power transformed data, supported by Bayesian Information Criterion for model selection, identified the 10 best age markers. The developed model was found to predict age with an absolute error of 2.1 years. The microarray results were confirmed for 4 markers on an independent cohort of 145 semen samples using pyrosequencing technology, and the whole set of 10 CpG sites will be further validated using amplicon bisulfite sequencing and Illumina technology. The study shows that epigenetic age prediction in semen is more demanding compared to age estimation in somatic cells and the higher number of CpG predictors is needed to achieve the desired level of prediction accuracy. This research has received funding from the EU Horizon 2020 VISAGE project (no. 740580).

630 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P549 - EPIGENETIC AGE ESTIMATION OF HEALTHY CZECH POPULATION BY AGEPLEX Helena Štefanová1, Barbora Blumová1, Veronika Holinková1, Lucie Kotková1, Rastislav Slavkovský1, Karolína Bartáková1, Jana Stránská1, Jiří Drábek1

1 Palacky University, IMTM- Faculty of Medicine and Dentistry, Olomouc, Czech Republic

Epigenetic (cytosine methylation) analysis of DNA gains momentum in forensic genetics because it can be used for authentication of natural DNA, tissue of origin typing, time of sample deposition estimation, revealing behavioural/physiological pattern of perpetrator (smoking, alcoholism, drug addiction, socioeconomical status, physical activity, body mass index, vege- tarianism, and age). Estimation of age has the direct application in investigative mode of forensic genetics: e.g. if applied during investigation of Barbora Němečková rape and murder in Kmetiněves, the Czech Republic, the 15 years old perpetrator Robin would have been found earlier than as a number 632 out of 700 tested in DNA dragnet. Also, epigenetic age estimation would help to distinguish true children among refugees that claim preferential treatment for underaged. Age profiling by cytosine methylation study was not yet performed in the Czech Republic. Thus, we extracted DNA from peripheral blood in EDTA of blood donors (n>200), performed bisulfita- tion reaction using EpiTect Fast Bisulfite kit and PCR followed by pyrosequencing with 5 markers (ELOVL2 C7, C1orf132 C1, TRIM59 C7, KLF14 C1, FHL2 C2) using AgePlex kit (QIAGEN) on PyroMark Q48. Age was calculated online at http://biovectis.com/forensic1/age-calculator webpage. Here we provide our results with descriptive statistics and discussion. Acknowledgements: Supported by NV16–32198A, CZ.02.1.01/0.0/0.0/16_013/0001674, TE02000058, IGA_LF_2019_003, and CZ.02.1.01/0.0/0.0/16_019/0000868.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 631 P550 - EPIGENETICS MEETS STR AND mtDNA IN FORENSIC GENETICS Jana Naue1,2

1 University Medical Center Freiburg, Institute of Forensic Medicine- Forensic Molecular Biology, Freiburg, Germany 2 University of Freiburg, Faculty of Medicine, Freiburg, Germany

The analysis of DNA methylation (DNAm) has gained a lot of attention especially for its applicability in age and tissue prediction. Regions of interest were mainly based on CpG sites represented in microarrays or connected to tissue-specific functions. However, DNAm analysis of common forensically investigated loci as autosomal short tandem repeats (STR) and the mitochondrial genome (mtDNA) has not yet been investigated as to its potential in forensic genetics. Analytical challenges are expected due to the repetitive structure of STRs which could be especially prone to damage during bisulfite conversion. Additionally, studies from other research areas provided controversial results regarding the presence of mtDNA methylation. However, age- as well as tissue-specific changes could be hidden in these areas, although tandem repeats normally keep a high DNAm level. The analysis of an informative DNAm pattern in combination with the STR allele or mtDNA haplotype on the same molecule could lead to a direct link between a trace source and the individual in a DNA mixture. In a first study, the DNAm of selected mtDNA and STR markers was investigated and analyzed in different tissues of deceased individuals using samples of bone, buccal swabs, blood, skeletal muscle, liver, brain and hair. The results demonstrate the challenge of correct mitochondrial DNAm analysis due to the circular structure and sequence context. DNAm was found to vary around STR regions. The pilot study also indicates tissue-specific DNAm differences e.g. in brain and shows successful STR genotyping from bisulfite converted DNA.

632 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P551 - EVALUATION OF HIRISPLEX-S SYSTEM MARKERS FOR EYE, SKIN AND HAIR COLOR PREDICTION IN AN ADMIXED BRAZILIAN POPULATION Leonardo Marano1, Jeppe Dyrberg Andersen2, Fernanda de Toledo Gonçalves3, Ana Laura Oliveira Garcia3, Cintia Fridman3

1 Laboratorio de Genetica Molecular Forense - Policia Cientifica do Parana, Divisão de Laboratorios, Curitiba, Brazil 2 University of Copenhagen, Department of Forensic Medicine, Copenhagen, Denmark 3 Universidade de São Paulo, Departamento de Medicina Legal, Ética Médica e Medicina Social e do Trabalho, São Paulo, Brazil

The inference of externally visible characteristics (i.e. skin, eye and hair color, height and facial features) from biological trace samples is known as Forensic DNA Phenotyping. The HIrisPlex-S system is a forensically validated tool for simultaneous eye, hair, and skin color prediction from DNA and has been reported to reach predictive power for skin colors, but investigations have mainly been carried out in homogeneous populations with minor admixture. In this study, we present the first evaluation of the HIrisPlex-S system in an admixed population. A total of 611 Brazilian individuals were genotyped for 39 out of the 41 HIrisPlex-S markers, distributed in 19 genes/gene regions (MC1R, TUBB3, SLC45A2, KITLG, LOC105374875, IRF4, TYR, OCA2, SLC24A4, HERC2, PIGU, LOC105370627, TYRP1, ANKRD11, BNC2, SLC24A5, ASIP, RALY and DEF8). The remaining 2 markers are being typed and will be added to the final analysis. The predictions of eye, hair, and skin color were carried out using the HIrisPlex-S prediction model (https://hirisplex.erasmusmc.nl/). The results were compared to the phenotypes for each individual, and the Area Under the Curve (AUC) for each phenotype feature was calculated using R statistics software (packages MASS, mlogit, ROCR, pROC and caret). AUC values were found to be higher for blue eye (0.88), brown eye (0.67), black hair (0.69), fair skin (0.70) and dark skin (0.70). Intermediate phenotypes reached lower values compared to those of the extreme phenotypes (light/dark). Our results demonstrate that the HIrisPlex-S system markers have great potential for use in admixed populations, including the Brazilian, but our results also demonstrate that the intermediate color groups are difficult to predict. Financial Support: FAPESP(16/03284–8), HCFMUSP/LIM40.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 633 P552 - EVALUATION OF SKIN-RELATED VARIANTS IN AFRICAN ANCESTRY POPULATIONS AND THEIR ROLE IN PERSONAL IDENTIFICATION Virginia Veltre1, Flavio De Angelis1, Gianfranco Biondi2, Olga Rickards1

1 University of Rome “Tor Vergata”, Biology, Rome, Italy 2 University of L’Aquila, Medicina clinica- sanità pubblica- scienza della vita e dell’ambiente, L’Aquila, Italy

Pigment-related genetic variants point out their role in personal identification as they can be considered predictors suitable for Forensic DNA Phenotyping (FDP) and mounting evidence suggest also their bio-geographic inferential power for gaining information about the individual geographical origin. The current research aims to explore the allelic status in several SNPs mapped in selected genes known to be involved in skin pigmentation: OCA2, HERC2, SLC45A2 and an intergenic region between BEND7/PRPF18. The genetic evaluation has been performed on 219 healthy people from African and African derived populations: Fon, Dendi, Bariba and Berba from Benin, Tuareg from Libya and Afroecuadorians. The genotypic results have been integrated with the available data from Phase 3–1KGP release and HapMap data in order to obtain a selected populations panel useful for their use as inferential model training set to test the likelihood of correct assignment to geographically differentiated human groups. Data reduction methods and two different classification algorithms based on Bayesian inference have been employed in order to compare the assignment likelihoods. Due to the close relationship between the environment and the color of somatic traits, these SNPs should also be considered suitable Ances- try Informative Markers. From this perspective, the proposed variants panel seems to properly interpret the geographic variation and some new interesting evidence could be pointed out in African mixed populations. The results support the use of phenotypic inference along with bio-geographical ancestry information as valid auxiliary tools in personal identification.

634 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P553 - EVALUATION OF THE HIRISPLEX-S SYSTEM IN A BRAZILIAN POPULATION SAMPLE Thássia Mayra Telles Carratto1, Letícia Marcorin2, Guilherme Debortoli3, Guilherme do Valle Silva1, Nadia Carolina Aguiar Fracasso2, Maria Luiza Guimarães de Oliveira2, Alison Luis Eburneo Pereira2, Amanda Beatriz Candelária da Silva1, Eduardo Antônio Donadi2, Aguinaldo Luiz Simões2, Erick da Cruz Castelli4, Heather L. Norton5, Esteban J. Parra3, Celso Teixeira Mendes Junior1

1 Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Departamento de Química, Ribeirão Preto, Brazil 2 Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Departamento de Genética, Ribeirão Preto, Brazil 3 University of Toronto at Mississauga, Department of Anthropology, Mississauga, Canada 4 Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Departamento de Patologia, Botucatu, Brazil 5 University of Cincinnati, Department of Anthropology, Cincinnati, USA

DNA Phenotyping is a technique that consists in predicting externally visible characteristics (EVC’s) of an individual using only its DNA genetic information. Nowadays, the HIrisPlex-S System, composed of 40 SNPs and a InDel, stands as a tested and validated tool capable of predicting eye, hair and skin color simultaneously. However, prediction of intermediate color phenotypes may be impaired in highly admixed populations, so the applicability of this tool in such populations must be evaluated. This study aims to analyze the HIrisPlex-S System accuracy in 278 individuals from Ribeirão Preto city and proximities, Southeastern Brazil. A total of 37 markers were obtained by Next-Generation Sequencing (NGS) and Infinium Multi-Ethnic Global-8 (MEGA) Kit Array (Illumina), while the 4 remaining markers were imputed using the MEGA Array data and the IMPUTE2 software. Predictions were performed in the HIrisPlex-S online tool. Parameters as AUC, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the model were calculated. Our report shows that the HIrisPlex-S proved to be a useful tool to determine extreme eye and hair pigmentation phenotypes, but fails in determining intermediate phenotypes accurately. This is a major drawback since intermediate phenotypes are common in highly admixed populations and the failure of the system in predicting these phenotypes hinder its use in these populations. Unfortunately, results obtained for skin color were not satisfactory in general (accuracy of 35.14 %). In conclusion, although the HIrisPlex-S System seems to be a good tool for forensic use in Europe, there is no evidence yet to support its use in highly admixed American populations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 635 P554 - EXAMINATION OF AGE-ESTIMATION MODEL FOR THE JAPANESE USING DNA METHYLATION SITES Xueting Guan1, Tsukasa Ohuchi1, Yui Igari1, Kiyotaka Usui1, Masaki Hashiyada2, Masato Funayama1

1 Tohoku University, Forensic Medicine, Sendai, Japan 2 Kansai Medical University, Legal Medicine, Osaka, Japan

Background and aims: Age estimation of unidentified persons plays a vital role in forensic investigations, and it is challenging. Recently, DNA methylation sites have been shown to be powerful markers to predict the chronological age, and several estimation models have been reported in some populations. To date, various statistical models have been analyzed separately. In addition, research on cadaver blood and the Japanese is scarce, the practical application of forensic age estimation requires additional researches. In this study, we analyzed 6 genes (ASPA, CCDC102B, C1orf132, ELOVL2, ITGA2B and PDE4C) using Japanese corpse blood and compared age-estimation models based on multivariate linear regression and quantile regression. Materials and methods: Genomic DNA was extracted from whole blood of 60 autopsied Japa- nese subjects (from 2 weeks to 91 years old), bisulfite conversion treatment was performed and the target regions were amplified by target PCR. The library preparation and massively parallel sequencing was carried out by the MiSeq system. The data were analyzed by CLC-Genomic Workbench (Qiagen). Statistical analysis and comparison of age-estimation models were performed in R v3.5.2 with R studio v1.1.463 using package quantreg v5.38. Results and conclusions: Using the most highly correlated sites from each gene, age-prediction models were constructed and compared. Consequently, it was suggested that the quantile regression model is suitable to precisely predict the actual age of humans by providing prediction intervals that change with increasing age, and with a smaller median average deviation of 7.76 years (linear based model: 8.09 years). It is planned to examine the validity of the quantile-regression based model for the Japanese.

636 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P555 - FORENSIC AGE PREDICTION WITH 25 NG OF INITIAL INPUT DNA USING BISULFITE PYROSEQUENCING Zhonghui Thong1, Jolena Ying Ying Tan1, Christopher Kiu Choong Syn1

1 Health Sciences Authority, Applied Sciences Group- DNA Profiling Laboratory, 11 Outram Road S169078, Singapore

Predicting the age of the donor of biological materials recovered from crime scenes can provide valuable leads in forensic investigations. The study of DNA methylation at age related CpG sites is currently regarded as the ‘gold’ standard for the prediction of age. Most studies with promising age prediction models, however, required several hundred ng of initial input DNA for bisulfite treatment prior to pyrosequencing for age prediction. While several studies have reported using more forensically relevant amounts of DNA at the PCR stage, the initial amount of input DNA used for bisulfite treatment was still high. In this study, we investigated the minimum amount of DNA required for bisulfite treatment using 27 individuals. To minimize the amount of DNA required for age prediction, we opted to predict age using only one locus (ELOVL2), instead of three loci (ELOVL2, TRIM59 and KLF14) reported in our earlier study. We compared the prediction models generated using stepwise multiple regression (SMR) analysis and multilayer perceptron (MLP) analysis. The age prediction model obtained by SMR comprising of one CpG predictor from ELOVL2 yielded a mean absolute deviation (MAD) of 4.09 years with 25 ng of DNA input. The MAD decreased to 3.51 years when two ELOVL2 CpG predictors were used. Analysis with MLP also yielded comparable MADs of 4.10 years and 3.37 years with one and two ELOVL2 predictors, respectively. With both SMR and MLP achieving more than 75 % correct prediction, prediction of age using two ELOVL2 CpG predictors at 25 ng of DNA input could be more applicable in the context of crime investigation where samples may be highly limiting.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 637 P556 - FORENSIC CogInfoCom CHALLENGES OF DNA‑PHENOTYPING Monika Nogel1, András Czebe1, Zsolt Pádár1, Gábor Kovács1

1 Széchenyi István University, Research Center for Forensic Sciences and Criminology, Győr, Hungary

Forensic science is highly focused on technological developments, especially with regard to forensic identification disciplines. Due to their support of the criminal justice system, DNA technologies have improved dramatically in recent decades. There is a range of new tools which can help law enforcement authorities to identify unknown suspects. Familial DNA searching through different databases could reveal familial or genealogical relationships. Moreover, one of the latest achievements of forensic genomic, DNA phenotyping (FDP) seeks to predict a person’s externally visible characteristics from biological samples. The use of FDP technologies has been characterized by various controversies. Databases and/or networks of databases, the information-gathering techniques require consideration of a technical, social and normative aspects for investigative purposes. It should, however, not be neglected that the introduction of FDP, necessitates thinking about its influence on human perceptions, decisions and interpretations as well. Analysis of big datasets require novel data-management tools and methods. Since genetic data are generally less transparent, bias and potential errors could be more or less obvious. If we apply forensic cognitive infocommunication (Forensic CogInfoCom) processes correctly, we are not faced with the risk that FDP technologies bias, instead of help the criminal justice system. The introduction of new DNA technologies is therefore accompanied by new Forensic CogInfoCom challenges as well.

638 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P557 - GENETIC INVESTIGATIONS OF BROWN EYED INDIVIDUALS WITH THE RS12913832:GG GENOTYPE Jeppe Dyrberg Andersen1, Maja Maria Birk Lunn1, Olivia Strunge Meyer1, Sara Beatriz Garcia1, Anne Bruun Kjærbye1, Niels Morling1, Claus Børsting1

1 Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V’s vej 11- 2100 Copenhagen, Denmark

The genetic basis for distinction between blue and brown eye colour primarily depends on one SNP, HERC2 rs12913832. Individuals with the genotypes rs12913832:AA and rs12913832:GA most likely have brown eye colours, whereas individuals with the genotype rs12913832:GG most likely have blue eye colours. However, approximately 3 % of Europeans with the rs12913832:GG genotype have brown eye colours. Here, we sequenced a total of 1 Mb in coding and upstream regions of SLC24A4, TYRP1, IRF4, SLC45A2, and OCA2 (which included parts of HERC2) in 40 Euro- peans with the genotype rs12913832:GG. Twenty-four individuals had the expected blue eye colour, and 16 individuals had brown eye colours. We found no variants in SLC45A2, IRF4, or the OCA2-HERC2 region to be associated with eye colour variation. However, 115 variants in TYRP1 and SLC24A4 were found to have a statistically significant association with both categorical eye colours (blue vs. brown) and quantitative eye colours (‘Pixel Index of the Eye’-score). As expected, many of the identified variants were located in haploblocks. The most promising variants were found in possible regulatory elements upstream of TYRP1 and SLC24A4. These findings suggest that both TYRP1 and SLC24A4 are involved in the formation of brown eye colours in individuals with the genotype rs12913832:GG.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 639 P558 - GLOBAL VARIABILITY AND PATTERNS OF LINKAGE DISEQUILIBRIUM IN THE OCA2-HERC2 REGION ASSOCIATED WITH HUMAN PIGMENTATION Philippe Suarez1, Diana Hall1

1 Unité de Génétique Forensique, Centre universitaire romand de médecine légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, Switzerland

The OCA2 gene is the key player in the pigmentation pathway. Mutations in this gene are responsible for albinism, and polymorphisms in and around OCA2 have also been associated with pigment variation. The OCA2 region is under positive selection in both Europeans and East- Asians. However, both associated variants and haplotypes favored by selection are different in each population. While the majority of OCA2 association studies have focused on SNP polymorphisms, very little is known about the genetic variability of several STRs present in this gene. Mutational mechanisms have an effect on linkage disequilibrium (LD) and data exist in support of a population genetic framework that combines patterns of variation of SNPs and STRs. In this report, we provide data on 12 STRs and 3 Indels spanning the OCA2-HERC2 region in 1,000 individuals from 52 populations around the world (HGDP–CEPH panel). Haplotypes were constructed integrating the newly typed markers with existing SNP data. For each individual, predictions of eye color previously published were also used for the analysis. Our preliminary data show that several STRs show atypical allele frequencies with one allele showing low frequency in Africa, East-Asia, Oceania and Native-America and striking high frequencies in Europe, Middle-East and Central-South Asia. These alleles show high LD values (r2 between 0.8 and 1) with surrounding SNP and STRs both at short- and long-range distance. In Europe, the haplotype most frequent in blue eye color individuals is different from the one most frequent in brown eye color individuals. Our data suggest that, STRs may show increased power in detecting genetic associations and may provide a finer characterization of the haplotypes and evolutionary events related to this genomic area.

640 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P559 - HERC2 (RS12913832) AND OCA2 (RS1800407) GENES POLYMORPHISMS IN RELATION TO IRIS COLOR VARIATION IN BELARUSIAN POPULATION Marina Shapturenko1, Svetlana Vakula1, Alexander Kandratsiuk1, Irena Gudievskaya2, Marina Shinkevich1, Arthur Luhauniou3, Sergei Borovko3, Alexander Kilchevsky1

1 Institute of Genetics and Cytology, Ecological genetics and biotechnology, Minsk, Belarus 2 Belarusian State Medical University, Ophthalmology, Minsk, Belarus 3 State Forensic Examination Committee, Forensic-biological examination, Minsk, Belarus

To develop specific predictive model of forensic case work for Belarusian population, we set up investigation the association between candidate pigmentation SNPs and quantitatively measured eye color. A total 627 individuals (380 female, 247 male) from Belarusian population were recruited for our study. All participants gave written informed consent before the study. Pig- mentation phenotypes were documented by photographs taken at ultra-close high-resolution shots (Canon 750d completed by macrocamera with circular flash-light). The color information of each image pixel was transformed from RGB (red-green-blue) to HSV (hue-saturation-value) triplet for digital classification of pheomelanin, eumelanin and non-pigmented iris areas based on developed color reference, in package Matlab (R2018a). To date 209 participants were genotyped for rs12913832 (HERC2) and rs1800407 (OCA2) through TaqMan approach. The SNP allele distribution in the target regions of individuals under research was shifted to the G-alleles for both spots: 79 % - rs12913832:G, 74 % - rs1800407:G. In accordance to the dominant model, brown eye color is the outcome in individuals with allele A (rs12913832:A>G). However, in our research this is not always for genotypes rs12913832:- GA – 37 % occurrences were detected with intermediate eye color. Further, we have gagged the effect of the HERC2-OCA2 variation on the light/dark iris color grade. High values OR (>30) for rs12913832 confirmed it strong relationship with iris color. The impact of rs1800407 (<5.2) was significantly weaker. The logistic regression revealed a low percentage (3.9 %) of incorrect predictions: 97.3 % of light-eyed individuals and 92.9 % of dark-eyed individuals were correctly classified through HERC2(rs12913832)/OCA2(rs1800407) model.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 641 P560 - IDENTIFICATION OF BLOOD-SPECIFIC AGE‑RELATED DNA METHYLATION MARKERS ON THE ILLUMINA METHYLATIONEPIC BEADCHIP Hussain Alsaleh1, Penelope Haddrill1

1 University of Strathclyde, Pure & Applied Chemistry, Glasgow, United Kingdom

The past decade has seen rapid development in DNA methylation (DNAm) microarrays, including the Illumina HumanMethylation27 and HumanMethylation450 (450K), which have played an essential role in identifying and evaluating age-related (AR) DNAm markers in different tissues. Recently a new array, the Illumina MethylationEPIC (~850,000 probes) was introduced, with nearly double the number of probes than the 450K. In this study, the newly added probes were tested for age association using a large blood cohort of 754 DNAm profiles assayed on Meth- ylationEPIC for individuals aged 0–88 years old. 21 novel AR CpG sites were discovered with Spearman’s abs(rho) >0.6 (P-value<10-83) and mapped to 17 genes, 9 (LHFPL4, SLC12A8, EGFEM1P, GPR158, PXN, TAL1, PDE1C, DUSP16, and FAM65C) of which never been reported before to be associated with age. The data was subsequently split into a 527-sample training set and a 227-sample testing set to build and validate two age prediction models using elastic net regression and multivariate regression. Elastic net regression selected 465 CpG markers with a mean absolute deviation (MAD) of 2.6 years based on the testing set. The top CpG markers with Spearman’s abs(rho) >0.6 were input into stepwise regression to select the best subset of markers for multivariate regression. The 16 selected CpG markers were linearly modelled with age, explaining 88 % of age-correlated variation in DNA methylation levels, and an age estimation accuracy of 3.8 years (MAD) based on the testing set. These results suggest MethylationEPIC probes for age estimation fall within the range of probes found on the previous Illumina platforms.

642 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P561 - IDENTIFICATION OF mRNA- AND DNA METHYLATION MARKERS FOR HUMAN AGE ESTIMATION BY MPS TECHNIQUES Katharina Hartmann1, Carolin Fix1, Ralf Horres2, Jens Amendt1, Richard Zehner1

1 Institute of Legal Medicine, University Hospital, Frankfurt am Main, Germany 2 GenXPro GmbH, Frankfurt, Frankfurt am Main, Germany

The human age estimation of living persons is relevant for administrative and criminal proceedings. Recently intensely debated was the need to determine the age of migrants without identification papers. Another forensically important application field is the age estimation of a defendant due to the age‑dependent penalty. Established methods for human age estimation may be unreliable, imprecise or even lead to detrimental health effects. Therefore many current studies describe molecular biological approaches for age estimation using mRNA- or DNA methylation markers. However, despite calculated mean absolute deviations (MAD) the variance within a certain age is too high to determine effective differences especially within the relevant age group around 20 years. In this study a genome wide C-methylation analysis by next generation sequencing was performed from blood samples. In addition a transcriptome analysis was carried out by Massive Analysis of cDNA Ends (MACE) in order to trace change of gene activity – also as a consequence of DNA‑methylation. We investigated blood samples from 19 persons aged 18 to 26 years and for comparison a clearly separated collective of 9 persons aged 47 to 54. Analyzing the C-methylation pattern and the corresponding transcriptome enable a detection of candidate loci within the genome which may be suited to discriminate within the age group between 18 and 26. By characterization of these sites, appropriate test systems will be generated and validated. The identified markers will be compared with the already published markers and their possible usability is discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 643 P562 - IDENTIFICATION OF NOVEL SPERM-CELL SPECIFIC EPIGENETIC AGE PREDICTORS USING AGILENT’S HUMAN DNA METHYLATION MICROARRAY Aleksandra Pisarek1, Ewelina Pośpiech1, Agata Jarosz1, Ana Freire-Aradas2, Anastasia Aliferi3, Marta Sikora-Polaczek4, Aneta Macur5, Michał Bochenek1, David Ballard3, Denise Syndercombe Court3, María Victoria Lareu2, Jarosław Janeczko5, Christopher Phillips2, Wojciech Branicki1,6

1 Jagiellonian University, Malopolska Centre of Biotechnology, Krakow, Poland 2 University of Santiago de Compostela, Forensic Genetics Unit, Institute of Forensic Sciences, Santiago de Compostela, Spain 3 King’s College London, Department of Analytical, Environmental and Forensic Sciences, Faculty of Life Sciences and Medicine, London, United Kingdom 4 Medical Centre Macierzyństwo, Medical Centre Macierzyństwo, Krakow, Poland 5 PARENS Fertility Centre, PARENS Fertility Centre, Krakow, Poland 6 Central Forensic Laboratory of the Police, Department of Biology, Warsaw, Poland

The discovery of the high predictive value of DNA methylation for an individual’s age led to extensive research aimed at validating markers and models in the field of forensic genetics. The availability of DNA methylation markers for somatic cells allowed the development of reliable DNA tests for individual age estimation based on analysis of blood and saliva. Because DNA methylation patterns of sperm cells are significantly different, these methods do not work for semen samples. In addition, few CpG markers that were proposed for sperm were selected by testing whole semen, which may also contain different amounts of other cell types. Therefore, sperm-specific CpG markers are still missing. To fill this gap we have conducted a discovery study that involved 12 semen samples from two extreme age categories (≤30 years vs. >40 years). DNA was extracted by means of differential extraction to remove the DNA fraction originating from somatic cells. The samples were subjected to microarray analysis using Agilent SurePrint G3 ChIP/CH3 Human Microarrays. The generated methylation data were analysed using Agilent Genomic Workbench 7.0.4.0 that enabled identification of 14 candidate CpG sites with P value > 0.01 and the relative methylation level difference between groups > 3 standard deviations. No- tably, these CpG sites were found to be located in genes involved in cell cycle regulation, maintaining genome stability and/or sperm cell functioning. In the next step the selected CpG sites will be validated using several independent sample sets from various European populations.

644 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P563 - IDENTIFICATION OF Y-CHROMOSOME METHYLATION MARKERS FOR MALE AGE ESTIMATION OF FORENSIC SAMPLES Rebecca Richards1, Jayshree Patel2, Kate Stevenson2, SallyAnn Harbison2

1 University of Auckland, School of Chemical Science, Auckland, New Zealand 2 The Institute of Environmental Science & Research Limited, Forensic Biology, Auckland, New Zealand

The use of DNA methylation markers to infer the chronological age of an unknown individual from a biological sample can provide important intelligence information to investigators. Current age estimation models are based on autosomal markers which are limited to single source samples. However, DNA mixtures are commonly found in forensic casework, particularly male-female mixtures in cases of sexual assault. Targeting Y-chromosome DNA methylation markers could allow for the age estimation of the male mixture component, without confound- ing information from the female component. In this study, we investigate the age predictive value of CpG markers found on the Y chromosome in three different biological fluids. We obtained publicly available Illumina Methylation Beadchip 450K microarray data for blood, saliva and semen samples from hundreds of male individuals of a wide age range. As DNA methylation is known to differ between biological fluids, we treated samples from each biological fluid as a separate dataset. Multiple feature selection methods (stepwise regression, variable importance and recursive feature elimination) were employed to identify subsets (n≤5) of the most statistically significant age-associated Y chromosome CpG markers. These subsets were then evaluated using different prediction models (multiple linear regression, random forest, support vector machine and Bayesian regularisation for feed forward neural networks). A mean absolute deviation (MAD) between true age and predicted age of ~4 years was obtained for semen, ~8 years for saliva and ~11 years for blood samples. Experimental corroboration of the predictive value for the identified novel candidate Y-CpG markers in a subset of individuals from the New Zealand population is currently underway.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 645 P564 - IMPLEMENTATION OF MASSIVELY PARALLEL SEQUENCING FOR FORENSIC DNA PHENOTYPING – RESULTS OF A WORLD-WIDE SURVEY Theresa Gross1, Peter Schneider1

1 University of Cologne- Faculty of Medicine and University Hospital Cologne, Institute of Legal Medicine, Cologne, Germany

This survey was part of the EU-funded research project VISAGE - VISible Attributes Through GE- nomics – (http://www.visage-h2020.eu) aiming to develop toolkits suitable for Forensic DNA Phenotyping by massively parallel sequencing (MPS). The purpose of this survey among forensic practitioners was i) To assess the level of implementation & use of MPS platforms plus their various forensic applications and ii) To identify the level of individual knowledge and previous practical experience as well as training and education needs in regard to Forensic DNA Phenotyping (FDP) – the DNA-based prediction of appearance (eye, hair and skin color), biogeographic ancestry and age. An online questionnaire with five different sections (Background information, Equipment & personnel, FDP applications, Education & Training, Final comments) and a total of 19 questions was designed. Invitations were sent out primarily to members of the ENFSI DNA Working Group and participants of the German DNA Profiling (GEDNAP) proficiency testing exercises. Preliminary results based on a response rate of 35 % from 31 different countries indicate a surprisingly high interest in MPS technology as 75 % of the participants either already own a MPS platform or are planning to purchase one within the next 2 years. Furthermore, 10 – 25 % of participants have already used FDP markers for forensic casework, either for criminal cases or for unidentified human remains. The final survey results will provide an in-depth overview on the overall interest and the current level of implementation of Forensic DNA Phenotyping and MPS instrumentation in forensic laboratories. This research received funding from the European Union’s Horizon 2020 Research and Innova- tion Programme under grant agreement number 740580 (VISAGE).

646 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P565 - IMPROVING ACCURACY OF AGE PREDICTION MODEL THROUGH MACHINE LEARNING ALGORITHM Zhonghui Thong1, Jolena Ying Ying Tan1, Eileen Shuzhen Loo1, Christopher Kiu Choong Syn1

1 Health Sciences Authority, Applied Sciences Group- DNA Profiling Laboratory, 11 Outram Road S169078, Singapore

The study of DNA methylation at age related CpG sites is currently one of the most promising methods used to predict age. Various statistical approaches ranging from simple linear regression to complex artificial neural networks (ANNs) have been used to correlate DNA methylation with chronological age. ANNs refer to groups of machine learning algorithms comprising layers of nodes that interact via carefully weighted connections to produce an outcome. Due to their adaptive learning process, ANNs can reduce the error during prediction output by systematically optimising the connective weights between the nodes within the network. We previously reported an age prediction model comprising of three CpGs using stepwise multiple regression (SMR). The present study aimed to further reduce prediction error from our previous work by employing multilayer perceptron (MLP), which is a class of ANNs. A total of 261 samples used previously were randomised to training and testing sets to reduce possible model overfit- ting. The mean absolute deviations (MADs) obtained from the training set were 3.78 years and 3.86 years using SMR and MLP, respectively. The SMR approach was subsequently validated with MAD of 4.08 years while the MAD for MLP was 3.87 years on the testing set. Although both SMR and MLP generated comparable MADs for both training and testing sets, MLP produced a smaller MAD difference between the two sets. Furthermore, the validated MADs for both approaches in this study were observed to be lower as compared to our previous study with validated MAD of 4.96 years, indicating the significance of sample randomization.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 647 P566 - INCORPORATING AND VALIDATING THE IMPACT OF PRIORS ON DNA PREDICTION OF EXTERNAL VISIBLE CHARACTERISTICS Maria-Alexandra Katsara1, Michael Nothnagel1, Manfred Kayser2, Wojciech Branicki3, Susan Walsh4, Hysi Pirro5, Ewelina Pośpiech6, Visage Consortium7

1 Cologne Center for Genomics- University of Cologne, Department of Statistical Genetics and Bioinformatics, Cologne, Germany 2 Erasmus MC University Medical Centre Rotterdam, Department of Genetic Identification, Rotterdam, Netherlands 3 Jagiellonian University, Kraków, Malopolska Centre of Biotechnology, Krakow, Poland 4 Indiana University Purdue University, Indianapolis IUPUI- Indianapolis- IN, Department of Biology, Indianapolis, United States Minor Outlying Island 5 St Thomas Hospital, Campus, Kings College, London, Department of Twin Research and Genetic Epidemiology, London, United Kingdom 6 Jagiellonian University- Kraków, Malopolska Centre of Biotechnology, Krakow, Poland 7 Erasmus MC University Medical Centre Rotterdam- Rotterdam, Department of Genetic Identification, Rotterdam, Netherlands

Predicting external visible characteristics (EVCs) from genetic data, often referred to as Foren- sic DNA phenotyping, has raised major interest in forensic genetics over the last years. Sev- eral studies have recently developed and forensically validated predictive tools comprising of labtools and statistical tools, for traits such as eye, hair and skin color. Using prior information, e.g. obtained from the trait prevalence across geographic regions or populations, may potentially improve prediction accuracy, but has not been investigated thus far. Here, we performed a systematic assessment of the impact of incorporating prior values on the prediction for a number of EVCs, including eye, hair and skin color, hair structure, male pattern baldness and freckles, in terms of commonly used performance measures and compared the model performance also to the outcome for a model without priors. We show that prediction is affected to a different degree for the different EVCs and also single EVC categories. We further show that, although priors have the potential to increase prediction performance, misspecification of prior values can lead to dramatic losses in overall accuracy. Our results emphasize the importance of a precise specifi- cation of priors in order to achieve valid and accurate results. This research has received funding from the EU Horizon 2020 VISAGE project (no. 740580).

648 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P567 - IS IT POSSIBLE TO DISTINGUISH BREAST-FEEDING FROM BOTTLE-FED INFANTS WITH microRNA PROFILING OF GASTRIC CONTENT? Yu Kakimoto1, Yutaka Matsushima1, Eriko Ochiai1, Motoki Osawa1

1 Tokai University School of Medicine, Department of Forensic Medicine, Isehara, Japan

Backgrounds: Recently we were consulted about a challenging case, where an infant died by poisoning and the mother insisted that she unintentionally gave the toxic drug through breast milk. Accordingly, we had to reveal whether the infant took breast milk or not. We first tried to elucidate the protein differences by immunoblotting, but the results were ambiguous, possibly because the recent artificial milk is quite similar to breast milk nutritionally. Then we aimed to utilize miRNA profiling of the gastric content (GC) for the differentiation between breast-feeding and bottle-fed infants. Methods: Breast milk (BM) samples were obtained from two mothers within a year after delivery. GCs of two breast-feeding infants (GBr), two bottle-fed infants (GBo), and two adults (GA) were sampled at autopsy. Total RNA was extracted from the supernatant after supernatant using miRNeasy mini kit. Small RNA libraries were prepared with TruSeq small RNA library prep kit, and sequenced in Illumina MiSeq. Read count was performed using htseq-count with miRBase v21. Coverage depth data were analyzed on the CLC genomics workbench. Results and Discussion: On average, 3.4 million reads were sequenced per sample, and totally 1,123 miRNAs were identified. Mapping rate of miRNA was 29 % and 16 % in BM and GC groups, respectively. GC groups shared the major miRNA profiling, including miR-192, miR-21 and miR- 141. Principal component analysis revealed that BM is distinguishable from GC groups, and relatively similar to GBr. Baggerley’s test indicated that miR-151a and miR-186 are significantly higher in BM and GBr than in GBo and GA. These data elucidate the stability of miRNAs in gastric content and the utility of miRNA profiling for the assessment of the last meal of the subject.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 649 P568 - MAPLEX - AN ANCESTRY-INFORMATIVE ASSAY FOR THE ASIA PACIFIC REGION Christopher Phillips1, Dennis McNevin2, Kenneth Kidd3, María de la Puente1,4, Catarina Xavier4, Robert Lagacé5, Sharon Wootton5, Mayra Eduardoff4, Ana Freire-Aradas1, Ana Mosquera Miguel1, Antonia Heidegger4, Daniel Power6, Sharif Akhteruzzaman7, Mark Barash2, Maria Corazon A De Ungria8, Theresa Gross9, Masaki Hashiyada10, Dadna Hartman11, Julianne Henry12, Sae Rom Hong13, Hwan Young Lee14, Carla Oz15, Elizabeth Peters16, AA Sajib7, NA Soliven8, Naomi Tuitoga16, Sheri Olson5, Lucy Dagostino6, Peter Schneider9, Walther Parson4,17, María Victoria Lareu1, Runa Daniel18,19

1 Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 2 Centre for Forensic Science, Faculty of Science, University of Technology Sydney, New South Wales, Australia 3 Department of Genetics, Yale University School of Medicine, Conneticut, Australia 4 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 5 Human Identification Division, Thermo Fisher Scientific, California, USA 6 Thermo Fisher Scientific, Melbourne, Victoria, Australia 7 Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka, Bangladesh 8 DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines, Quezon City, Philippines 9 Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany 10 Department of Legal Medicine, School of Medicine, Kansai Medical University, Kansai, Japan 11 Forensic Services, Victorian Institute of Forensic Medicine, Victoria, Australia 12 Research- Education and Training, Forensic Science SA, South Australia, Australia 13 Department of Forensic Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea 14 Department of Forensic Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea 15 Forensic DNA Laboratory, Division of Identification and Forensic Science (DIFS)- Israel Police-National H.Q., Jerusalem, Israel 16 Forensic Biology & DNA Laboratory, Forensic Science Services, Fiji Police Force, Fiji 17 Forensic Science Program, The Pennsylvania State University, PA, USA 18 Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Victoria, Australia 19 National Centre for Forensic Studies, Faculty of Science and Technology, University of Canberra, Australian Capital Territory, Australia

Forensic DNA Intelligence has rapidly emerged as an valuable investigative tool. The ability to estimate the biogeographical ancestry (BGA) of the donor of an evidential sample enables more efficient use of valuable police and forensic resources and assist in decision making in the primary stages of an investigation. Application of massively parallel sequencing (MPS) has greatly expanded the potential for intelligence capabilities. An MPS-based ancestry informative marker panel analysing 164 sequences constructed with a specific focus on the differentiation of populations from South Asia, East Asia and Oceania in addition to differentiating between Africans, Europeans and indigenous Americans. The panel combines 112 established binary SNPs with two highly informative SNP-based marker sets; 32 multi-allelic SNPs (tri-and tetra-allelic loci) and 20 microhaplotypes consisting of 73 SNPs [1].

650 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS A trial of this panel was conducted involving laboratories in the Asia Pacific and the Middle Eastern regions. Participating laboratories contributed population samples of known ancestry (n=50). In addition, to ancestry predictive performance and the development of reference population data sets, this study reports the component marker characteristics and panel optimisation indicating high reproducibility and high sensitivity with full profiles achievable using 125pg of DNA. STRUCTURE v2.3.4 analysis of the population reference set indicates differentiation of at least seven global populations: sub-Saharan Africa, Middle East, Europe, South Asia, East Asia, Oceania and America using MAPlex. Current work is focused on differentiating East Asia and South East Asia. 1.Kidd K., et al Evaluating 130 Microhaplotypes across a Global Set of 83 Populations, Forensic Sci Int Genet, 2017.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 651 P569 - NEW MULTIPLEX STRATEGY FOR DNA METHYLATION- BASED AGE PREDICTIONS FROM LOW AMOUNTS OF DNA VIA PYROSEQUENCING Jan Fleckhaus1, Markus A. Rothschild1, Peter Schneider1

1 Institute of Legal Medicine, Faculty of Medicine- University of Cologne, Cologne, Germany

DNA methylation-based age prediction is a promising new tool for forensic molecular biology. Over the last years, a considerable number of age-dependent methylated “age CpGs” has been examined. There is growing understanding of their performance in different tissues and in the presence of various pathogenic factors and environmental conditions. Less focus has been put on technical demands of the method such as the required amount of input DNA which is particularly relevant in a forensic casework setting. Quantitative methylation analysis should be performed from at least 10ng bisulfite converted DNA which requires sensitive sequencing methods. Pyrosequencing is such a sensitive method, but the common workflow requires independent PCR and sequencing reactions for different target regions. The amount of input DNA thus has to comply with the number of target regions included in the applied age prediction model. To overcome this limitation, we developed a multiplex strategy including a multiplex pre-amplification of seven target regions with 14 age CpGs associated with the genes of ELOVL2, CCDC102B, C1orf132, KLF14, EDARADD, PDE4C and SST. By analyzing ten converted DNA samples in triplicates both with the new multiplex and the standard singleplex strategies we were able to directly compare the two approaches. The observed methylation values displayed no significant differences for eight and only small but significant differences for six of the 14 investigated CpG sites between the two analytical strategies. No precision differences were observed. Our results indicate that the multiplex strategy could act as an alternative for age predictions in cases when only limited amounts of DNA are available. For confirmation of these results larger datasets need to be investigated.

652 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P570 - OXFORD NANOPORE SEQUENCING AS A GOOD STRATEGY TO STUDY DNA VARIATION OF THE ENTIRE MC1R GENE Ewelina Pośpiech1, Agata Jarosz1, Magdalena Kukla-Bartoszek2, Magdalena Zubańska3, Agnieszka Bronikowska4, Magdalena Spólnicka5, Wojciech Branicki1,5

1 Jagiellonian University in Krakow, Malopolska Centre of Biotechnology, Krakow, Poland 2 Jagiellonian University in Krakow, Faculty of Biochemistry, Biophysics and Biotechnology, Krakow, Poland 3 Police Academy in Szczytno, Institute for Security Sciences Faculty of Security and Legal Sciences, Krakow, Poland 4 Collegium Medicum of the Jagiellonian University, Department of Dermatology, Krakow, Poland 5 Central Forensic Laboratory of the Police, Biology Department, Warsaw, Poland

Advances in high-throughput sequencing and big data analysis methods significantly expanded the opportunities for predictive DNA analysis in forensics. Red hair colour was the first physical appearance phenotype considered for forensic DNA intelligence purposes, mainly because of high visibility and a simple quasi-Mendelian inheritance pattern of this trait. The MC1R gene responsible for most of the cases of red hair colour, affecting other hair colours and skin colour, comprises a single exon that shows very high level of allelic heterogeneity. The HIrisPlex-S model most commonly used to predict pigmentation traits includes data for only the 11 most common MC1R variants while it is clear that the analysis of the entire MC1R sequence is necessary to accurately predict pigmentation traits. In this research we show that the Oxford Nanopore Technology offers a good alternative to study the sequence variation of the entire MC1R gene. MinION was used to analyse the 1590 bp MC1R exon and promoter in a cohort of 126 individuals including 65 red-haired participants. Three sequencing runs were conducted acquiring on average >18 000 reads/sample/MC1R sequence. Bioinformatics pipeline included Albacore for basecalling and demultiplexing, NanoOK for quality metrics, Minimap2 for mapping and Nanopolish for variants calling. Assigned DNA variants were validated using Ion Torrent technology provided with Ion Xpress Plus Fragment Library Kit and PGMTM machine. Systematic errors were successfully sorted out based on Nanopolish quality score, coverage and allele frequency. The study proves the Nanopore Sequencing to be a suitable solution for the entire MC1R gene sequence assignment that paves the way for the improved prediction accuracy of pigmentation in forensic investigations.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 653 P571 - PERCEPTION OF BLUE AND BROWN EYE COLOURS Olivia Strunge Meyer1, Claus Børsting1, Jeppe Dyrberg Andersen1

1 Section of Forensic Genetics- Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V’s vej 11- 2100 Copenhagen, Denmark

Eye colour is a prominent visible trait in Europeans and extensively studied in forensic genetics. There is a range of different eye colours varying from light blue to dark brown. Multiple methods for quantitative measurements and categorization of eye colour has been presented. However, knowledge on the agreement between categorization methods and the general perception of eye colour is lacking. In order to use prediction of eye colour in forensic cases, the perception of eye colour needs to be understood. Here, we categorized eye colour based on the subjective evaluation of eye colour by 30 Danes. Each participant evaluated 466 digital photos of eyes, 24 of which were shown to the participants twice. They were asked to categorise eye colour in a three-category and a two-category system. The three-category system consisted of the categories blue, intermediate, and brown. The participants were asked to further categorize the intermediate eye colours in either intermediate and blue (final category blue) or intermediate and brown (final category brown), which created the two-category system. We found that the two-category system corresponded best with the perception of eye colour. Disagreements in the eye colour categorization by at least 10 % of the participants were observed for 10 % of the pictures using the two-category system. The genetic basis for distinction between blue and brown eye colour primarily depends on a single SNP, rs12913832. This SNP is included in the Pre- cision ID Ancestry panel used for ancestry inference. Likelihood ratios based on the genotype of rs12913832 and the two-category system were: LR=P(rs12913832:GG blue)/P(rs12913832:GG brown)=10, LR=P(rs12913832:AA brown)/P(rs12913832:AA blue)=54, and LR=P(rs12913832:GA│brown)/P(rs12913832:GA│ blue)=19. │ │ │ │

654 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P572 - PHENOTYPE PREDICTION ACCURACY – A SWEDISH PERSPECTIVE Klara Junker1, Adam Staadig2,3, Maja Sidstedt1,4, Andreas Tillmar2,3, Johannes Hedman1,4

1 National Forensic Centre, Swedish Police Authority, Linköping, Sweden 2 Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden 3 Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden 4 Applied Microbiology- Department of Chemistry, Lund University, Lund, Sweden

Methods for SNP-based phenotype prediction have been developed, but prediction accuracy data from several populations and regions are missing. We analyzed the accuracy of hair and eye colour predictions for 111 individuals residing in Sweden, using the MiSeq FGx Forensic Ge- nomics System (Verogen). We compared prediction accuracy using the suggested probability threshold of ≥ 0.7 to using the highest probability value. The proportions of correct predictions were overall equivalent with or without threshold, but when a ≥ 0.7 threshold was applied, 12 % of eye colour predictions and 62 % of hair colour predictions were excluded. Below, we present results based on the highest probability value. Half of our cohort had blue eyes, one third brown and the rest intermediate/green. Predictions of brown eye colour were correct in 84 % of the cases, compared to 78 % of blue eye predictions. Intermediate/green eyes were never predicted. Individuals with incorrect brown eye predictions had either blue or green eyes, whereas individuals with incorrect blue eye predictions had green eyes, never brown. Half of our cohort had brown hair, a third blond, a tenth black and the remaining 6 % red. Predictions of red hair colour was correct in 83 % of the cases, compared to 72 % for brown, 56 % for black and 50 % for blond. Individuals with incorrect predictions of blond hair typically had brown hair, never black. Individuals incorrectly predicted to have brown hair had either black or blond hair. In summary, eye colour predictions were often correct, but the system failed to predict intermediate/green eye colour in our cohort. Hair colour prediction accuracy was higher for some colours than others, with discrepancies usually related to prediction or observation of brown hair.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 655 P573 - PREDICTION AND VALIDATION OF EYE COLOR ACROSS IRANIAN POPULATION WITH A SINGLE BASE EXTENSION GENOTYPING ASSAY Sayed Mostafa Hosseini1, Mahmoud Tavallaei1, Alireza Rafati1

1 Kawsar Human Genetics Research Center- 41 Majlesi St.- Vali Asr St.- Tehran- Iran, Forensic genetic, Tehran, Islamic Republic of Iran

Aim: To evaluation of six informative single nucleotide polymorphism (SNP) markers which associated with the prediction of human eye color in 106 individuals of Iranian population with different ethnic group. Methods: In this cross-sectional study, 2 mL blood samples from 106 healthy and unrelated individuals with informed consent were collected at the Ophthalmology laboratory of the Depart- ment of Human Genetics, the Baqiyatallah Medical Science University between 2015 to 2017. A Digital photographic image of their iris was taken simultaneously with a macro lens, ensuring that light conditions and similar distance were utilized for each photo for standardization. We analyzed six informative SNPs (rs12896399, rs16891982, rs1393350, rs12203592, rs12913832, rs1800407) with SNaPshot assay. The multinomial regression prediction model was used for these informative SNPs, according to which we calculated sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the area under the receiver characteristic operating curves (AUC) to increase accuracy of the eye color prediction. Results: Brown eye color was observed in 20 %, blue in 30 % and intermediate in 50 % participants. Through statistical analysis of logistic regression, the AUC value of IrisPlex markers was evaluated to determine eye color. The AUC values for dark eyes and blue eyes were 0.99 ± 0.0004 and 0.949 ± 0.0100, while for intermediate eye colors, the value was 0.9505 ± 0.017. Conclusions: IrisPlex as a useful tool is capable of rapid application in practical forensic genetic casework. However, the IrisPlex markers alone were not sufficient, and there is a need to include additional markers to increase the accuracy of eye color prediction.

656 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P574 - QBICO: A NOVEL LAB TOOL FOR THE COMBINED ACCURATE ASSESSMENT OF BISULFITE-CONVERTED DNA QUALITY AND QUANTITY IN FORENSIC EPIGENETIC APPLICATIONS Athina Vidaki1, Vivian Kalamara1, Manfred Kayser1

1 Erasmus MC University Medical Center, Genetic Identification, Rotterdam, Netherlands

Differential DNA methylation profiling has been introduced for forensic applications like age estimation. Various targeted techniques are employed involving bisulfite conversion, which significantly fragments DNA, while DNA recovery and conversion rates are highly variable. Thus far, no method exists to assess the DNA quality/quantity after bisulfite conversion, which impacts all downstream analyses. Here, we developed the first of its kind lab tool, a 5plex TaqMan-based quantitative PCR assay, to assess a sample’s bisulfite DNA recovery, conversion rate, fragmentation and inhibition. Two primer/probe sets target a single-copy gene via a short (85 bp) and long (235 bp) PCR fragment, allowing to estimate bisulfite-converted DNA concentration and fragmentation. Two primer/probe sets target the genomic (118 bp) and converted version (148 bp) of a highly multi-copy locus, allowing to estimate a genome-wide bisulfite conversion efficiency. One primer/probe set targets a spiked-in artificial DNA (99 bp), allowing to estimate PCR inhibition. The lab tool was developed using the EpiTect MethyLight kit (Qiagen) and optimized using synthetic DNA standards (IDT) (Efficiencies=90–100 %, R2=>0.99). To assess its performance we performed forensic developmental validation (e.g. sensitive: 97pg, repeatable: Mean SD:0.058, specific: human). Further, we applied this new lab tool to 4 DNA amounts (100, 10, 1, 0.1ng) from 3 DNA samples using bisulfite conversion kits from 7 companies. Depending on the starting quantity, bisulfite DNA recoveries ranged from 8.5–100 %, conversion efficiencies from 78–99.9 %, while certain kits highly fragmented DNA. We propose qBiCo for standardizing forensic epigenetic analyses. This research received funding from the EU Horizon 2020 VISAGE project (no. 740580).

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 657 P575 - SCHIZOPHRENIA AND EPIGENETICS IN FORENSIC SCIENCES Filiz Ekim Cevik1

1 Istanbul University Cerrahpasa, Institute of Forensic Sciences- Department of Medicine, Istanbul, Turkey

Epigenetic modifications are one of the most important heritable alterations in the DNA. Epi- genetics is also related in the forensic field. The interest in forensic epigenetic have lately been increasing significant.The DNA methylation level across the genome, as a response to various brief or prolonged environmental stimuli, result in individual epigenomic variations referred to as epigenetic fingerprint. Modern advances in the field of epigenetics especially can discriminate various single-source DNA samples such as bodily fluids collected from the crime scene. The beginning of DNA methylation studies in forensic science is fundamental to support the conventional STR profiling. It is quite evident that in near future this technique may be utilized to produce independent evidence as well as corroborative evidence to detect, and solve an array of forensic cases. The epigenome expressed by human behavior is dynamic in nature. It can modified with internal such as embryonic as well as external factors such as environmental. This is why epigenome data interpretation is more challenging than genomic data. We interest on schizophrenia because it is the illness most strongly related with criminal behavior. We hy- pothesize that schizophrenia is associated with a combination of genes and altering the effects of environmental influence on the brain and genes. Epigenetics of schizophrenia provides important information on how the environmental factors affect the genetic structure of the disease. DNA methylation plays a pivotal role in etiology for schizophrenia. The process will be highly promising in the court of law. In this review will present an overview of recent research on the relationship between schizophrenia, epigenetics.

658 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P576 - STABILITY OF DNA METHYLATION STATUS OVER THREE DIFFERENT DAYS Olivia Holländer1, Kristina Schwender2, Heidi Pfeiffer1, Marielle Vennemann1

1 University of Münster, Institute of Legal Medicine, Münster, Germany 2 University of München, Institute of Legal Medicine, München, Germany

DNA methylation has recently been introduced as a predictor of chronological age of a person or donor of a trace. Buccal swabs are of particular importance because they provide a non-invasive and comfortable method to harvest cells. In this study the time-dependent variation/ stability of individual DNA-methylation status were investigated. DNA methylation of 4 CpG sites (PDE4C, SST, EDARADD and KLF14) was analyzed in numerous individuals over 3 different days, to determine whether DNA methylation stays the same over different days or differs. The analysis was performed using a minisequencing multiplex reaction (SNaPshot, Thermo Fisher) and pyrosequencing (PyroMark Q48, Qiagen). Age prediction was performed using a model from a previous study (separate poster presentation). The observed stabilities of DNA methylation status will be discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 659 P577 - THE DEVELOPMENT OF EPIGENTIC METHYLATION FOR APPLICATIONS IN BODY FLUID IDENTIFICATION AND PHENOTYPING Bruce Mccord1

1 Florida International University, Department of Chemistry and Biochemistry, Miami, USA

Over the past few years we have been working to develop a forensic methodology for the determination of a suspects body fluid type and phenotype using a set of epigenetic loci that can be easily implemented into forensic casework. During this time our group has identified a wide variety of singleplexed epigenetic markers for use in body fluid identification, age determination and smoking status. These markers include loci suitable for the identification of blood, semen, saliva, and vaginal epithelia. The markers are highly stable and have been shown to produce accurate results for 20 year old samples. The quantitative aspects of this work permit us to determine not only the identity of the body fluid, but its relative amount in simple mixtures of blood and semen. Moreover, we can use this same process to define a suspects age and smoking status. In our project we utilize whole genome data from chip arrays to identify potential sites for epigenetic biomarkers. These locations are then amplified and checked for methylation status. Next a set of CpGs are amplified and examined to determine correlation with the desired body fluid or phenotype using pyrosequencing. If the loci are sufficiently discriminatory, we also can develop assays which rely on real time PCR. Most recently we have been developing a body fluid multiplex in order to conserve sample. At present we have developed this multiplex for body fluid identification using pyrosequencing and are extending this work to MPS platforms. The results from our study indicate that epigentic genotyping can provide important information to aid in criminalistics, particularly in situations in which body fluid identification or age determination is necessary at the trace level or post DNA extraction.

660 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P578 - THE EYELID TRAIT ASSOCIATED SNPs INVESTIGATION IN CHINESE HAN ADULTS Qian Wang1, Bo Jin2, Xiaoying Luo1, Zhilong Li1, Youjing Jiang1, Weibo Liang1, Lin Zhang1, Meili Lv3

1 Sichuan University, Department of Forensic Genetics- West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 North Sichuan Medical College, Department of Forensic Medicine, Nanchong, China 3 Sichuan University, Department of Immunology, West China School of Basic Medical Sciences and Forensic Medicine, Chengdu, China

DNA-based prediction of facial morphology could contribute to description of an unknown person and forensic reconstruction of physical appearance in the field of forensic DNA phenotyping (FDP). Eyelid trait represents one of the most distinguishing facial phenotypes in East Asians. Several SNPs associated with eyelid trait including single and double eyelids have been suggested in previous reports [1–2]. Here, a primary investigation was performed on another eyelid trait, i.e. epicanthus. A total of 723 Chinese Han adults were included in the simulated case-control study, which was divided into with epicanthus (316 cases) and without epicanthus (407 cases) and four SNPs were genotyped using PCR-RFLP. Results of association test showed that a significantly reduced with epicanthus incidence was observed in the rs2738265 GG genotype compared with CC genotype (OR = 0.60, 95 %CI = 0.37–0.97). Furthermore, reduced with epicanthus incidence was also revealed to be associated with the CG/GG genotypes of rs2738265 (OR = 0.67, 95 %CI = 0.47–0.96). Further study including more samples is recommended to discover additional determinants of eyelid trait. 1. B. Jin, J. Zhu and H. Wang, etc: A primary investigation on SNPs associated with eyelid traits of Chinese Han Adults. Forensic Science International: Genetics Supplement Series. 2015; 5: e669-e670. 2. Q. Wang, B. Jin and XY. Luo, etc: Association between BMP4 gene polymorphisms and eyelid traits in Chinese Han population. Forensic Science International: Genetics Supplement Series. 2017; 6: e355-e356.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 661 P579 - TIME FRAME PREDICTION FOR THE DEPOSITION OF A BIOLOGICAL SAMPLE USING CLOCK GENE EXPRESSION PATTERNS Lorena Girón-Santamaría1, Ana Freire-Aradas1, María de los Ángeles Casares de Cal2, Antonio Gómez-Tato2, Christopher Phillips1, María Victoria Lareu Huidobro1, Ana Mosquera Miguel1

1 Institute of Forensic Science, University of Santiago de Compostela, Forensic Genetic Unit, Santiago de Compostela, Spain 2 Faculty of Mathematics, University of Santiago de Compostela, Santiago de Compostela, Spain

The continuous improvement of the forensic genetic analysis brought substantial improvements in the information obtained from even minimal amounts biological material, which can provide police with valuable investigative leads. Determining the time-of-day at which a sample was deposited could yield key information relating to potentially unresolved questions: 1) estimating the time the crime was committed, 2) estimating the period of time elapse from when someone was at the crime scene, 3) helping to establish police investigation hypothesis, and 4) reducing the suspect pool by excluding those with time-related alibis. Our study focused on selection of mRNAs in CLOCK genes and CLOCK controlled genes: loci playing a core role in the regulation of circadian rhythms. Compilation of CLOCK mRNAs forms the first step in developing forensic tests to predict the time-of-deposition (ToD) of a sample. We analysed the biggest dataset so far made (50 healthy donors) and chose to test saliva, often encountered in crime scenes but not widely studied for ToD. Saliva was collected at set times of the day (every 2 and 4 hours), and gene expression levels of in PER1; PER3; CRY1; TRIB1; MKNK2; and REV-ERBα CLOCK genes; plus ACTB reference gene, were quantitatively measured by RT-qP- CR. Circadian rhythm expression was evaluated accounting for differences in gene expression levels at different time points in 24-hour cycles. Although considerable variability was observed in gene expression levels, a prediction model has now been developed to estimate the most probable time frame for sample deposition in saliva, and this forms the next stage in our evaluations of the developed ToD tests.

662 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P580 - VALIDATED INFERENCE OF SMOKING HABITS FROM BLOOD WITH A FINITE DNA METHYLATION MARKER SET Silvana C.E. Maas1,2, Athina Vidaki1, Roy Wilson3, Alexander Teumer4,5, Fan Liu1,6,7, Joyce B.J. van Meurs2,8, André G. Uitterlinden2,8, Liesbeth Duijts9, Vincent W.V. Jaddoe2,10,11, Sonja Kunze3, Annette Peters3,5,12, M. Arfan Ikram8, Hans J. Grabe13, Janine F. Felix2,10,11, Melanie Waldenberger3,5, Oscar H. Franco2, Mohsen Ghanbari2,14, Manfred Kayser1, Bios Consortium2

1 Erasmus MC University Medical Center, Genetic Identification, Rotterdam, Netherlands 2 Erasmus MC University Medical Center, Epidemiology, Rotterdam, Netherlands 3 German Research Center for Environmental Health, Epidemiology, Munich, Germany 4 University Medicine Greifswald, Community Medicine, Greifswald, Germany 5 Germany Center for Cardiovascular Research, Partner Site Greifswald, Greifswald, Germany 6 University of Chinese Academy of Sciences, Key Laboratory of Genomic and Precision Medicine, Beijing, China 7 Chinese Academy of Sciences, Beijing Institute of Genomics, Beijing, China 8 Erasmus MC University Medical Center, Internal Medicine, Rotterdam, Netherlands 9 Erasmus MC University Medical Center, Pediatrics- Division of Neonatology, Rotterdam, Netherlands 10 Erasmus MC University Medical Center, Pediatrics, Rotterdam, Netherlands 11 Erasmus MC University Medical Center, The Generation R Study Group, Rotterdam, Netherlands 12 Ludwig-Maximilians-Universitat Munich, Medical School, Munich, Germany 13 University Medicine Greifswald, Psychiatry and Psychotherapy, Greifswald, Germany 14 Mashhad University of Medical Science, Genetics, Mashhad, Islamic Republic of Iran

Tobacco smoking impacts epigenetic variation. Besides applications in epidemiology and public health, inferring a person’s smoking habit from blood is particularly relevant in forensics to provide investigative intelligence information in cases without known suspects. We envision an extended concept of forensic DNA phenotyping covering epigenetic prediction of life style factors and age in addition to genetic prediction of appearance and ancestry. We employed previous epigenome-wide association studies for marker discovery and used data from six population-based cohorts (N=3,764) for model building. This allowed us to identify a finite set of 13 CpGs most suitable for inferring smoking versus non-smoking status from blood, achieving a cumulative Area Under the Curve (AUC) of 0.901. Internal five-fold cross-validation yielded an average AUC of 0.897±0.137. External model validation in an independent population-based cohort (N=1,608) achieved an AUC of 0.911. The same 13 CpGs provided accurate inference of current (average AUCcrossvalidation 0.925±0.021, AUCexternalvalidation0.914), former (0.766±0.023, 0.699) and never smoking (0.830±0.019, 0.781) status from blood. Model application to children revealed highly accurate inference of the true non-smoking status from blood drawn at six (accuracy 0.994, N=355) and ten (0.994, N=309) years of age, implying that prenatal smoking exposure having no impact on the model’s accuracy when being applied to adults. The finite set of DNA methylation markers and the validated statistical model introduced here allow reliable and accurate inference of a person’s smoking habit from blood, which we envision becoming useful in future forensic applications, provided that a sensitive lab tool is developed and forensically validated.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 663 P581 - VICTORIA’S UNIDENTIFIED HUMAN REMAINS – WHAT MORE CAN WE DO TO IMPROVE MISSING PERSON’S INVESTIGATIONS? April Stock1, Michelle Spiden1, Dadna Hartman1,2

1 Victorian Institute of Forensic Medicine, Molecular Biology, Southbank, Australia 2 Monash University, Department of Forensic Medicine, Southbank, Australia

The investigation of missing persons in Victoria, Australia, is a multi-disciplinary approach reliant on the co-operation of different agencies. Victoria Police hold ante-mortem information, including the physical description of the missing person, as well as any dental/medical records and fingerprint information; whilst the post-mortem data is collected and stored by the Victorian Institute of Forensic Medicine. Our laboratory is responsible for the DNA information pertain- ing to these cases. We utilise both nuclear DNA analysis (via STR multiplex) and mitochondrial DNA (via Sanger sequencing) to perform kinship and direct comparisons to reference profiles. The expanding body of work on phenotypic and ancestry SNPs, and the growing acceptance of Massively Parallel Sequencing, will soon see port-mortem reports for unknown deceased containing information mirroring the personal descriptors in missing persons reports, such as hair/ eye colour and ancestry. This study details the application of the IonAmpliSeq™ PrecisionID Panel (Thermo Fisher) on 29 unidentified remains cases, with the aim to assess the phenotypic and ancestry inferences, as well as the effectiveness of the AmpliSeq™ PrecisionID Panel and IonTorrent workflow on degraded samples. Using the phenotypic predictive tool Hirisplex and the IonTorrent analysis software (Thermo Fisher), we were able to achieve full phenotypic and ancestry results for 14 samples with DNA yields as low as 0.006ng/ul. Furthermore, a comprehensive national missing persons investigative capability – the National Missing Persons Victim System (NMPVS) coupled with DNA kinship – has recently come online across Australia and companies offering genealogy services could assist with the investigation of our long term unidentified human remains.

664 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P582 - WHAT MAKES YOUR “EYES” LOOK DIFFERENT? Qian Wang1, Bo Jin2, Chuanqi Zhang2, Li Li2, Zhilong Li1, Youjing Jiang1, Xiaoying Luo1, Lin Zhang1, Weibo Liang1, Yanyun Wang3

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 North Sichuan Medical College, Department of Forensic Medicine, Nanchong, China 3 Sichuan University, Laboratory of Molecular Translational Medicine- Center for Translational Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second University Hospital, Chengdu, China

Forensic DNA phenotyping (FDP) aims to predict the appearance traits of unknown sample donors based on DNA, providing investigative leads for police in the absence of a suspect. Facial morphology is one of the most significant appearance traits, therefore it represents a promising subfield of FDP. In our previous studies, several SNPs associated with eyelid trait were found [1– 2]. Here, we tried to figure out if there are any genetic markers associated with the appearance of the eyes besides eyelids. Palpebral fissure was selected and the ratio of palpebral fissure width to height (RPF) was divided into two categories, i.e. low ratio (1.8–2.4) and high ratio (3.6–5.4). A simulated case-control study was conducted in 156 Chinese Han adults including low RPF (92 cases) and high RPF groups (64 cases) and two SNPs were genotyped using SNaPshot® Mul- tiplex Kit. Significantly increased incidence of high RPF was associated with the AG genotype of rs2074612 compared with GG or GG/AA genotypes (AG versus GG: OR = 2.36, 95 %CI = 1.08– 5.18; AG versus GG/AA: OR = 2.07, 95 % CI = 1.03–4.16). Significantly increased incidence of high RPF was also observed in the rs2074612 AG/AA genotypes (OR = 2.14, 95 %CI = 1.01–4.53). No genotype distribution difference was found for rs2071047. Further study including more samples is needed to discover more SNPs associated with palpebral fissure trait. 1. B. Jin, J. Zhu and H. Wang, etc: A primary investigation on SNPs associated with eyelid traits of Chinese Han Adults. Forensic Science International: Genetics Supplement Series. 2015; 5: e669-e670. 2. Q. Wang, B. Jin and XY. Luo, etc: Association between BMP4 gene polymorphisms and eyelid traits in Chinese Han population. Forensic Science International: Genetics Supplement Series. 2017; 6: e355-e356.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 665 P583 - Y-CHROMOSOME BASED EPIGENETIC AGE ESTIMATION IN BLOOD: A NOVEL APPROACH FOR ESTIMATING AGE OF MALES FROM MALE-FEMALE DNA MIXTURES Athina Vidaki1, Diego Montiel González1, Benjamin Planterose Jiménez1, Manfred Kayser1

1 Erasmus MC University Medical Center, Genetic Identification, Rotterdam, Netherlands

Inferring an unknown individual’s age from crime scene traces can provide important leads for police investigations in search for unknown perpetrators unidentifiable with forensic DNA profiling. Current attempts on age estimation using DNA methylation are all based on autosomal markers, and thus, not suitable for male-female DNA mixtures commonly found in criminal casework. Targeting male-specific Y-chromosomal DNA methylation markers informative for age would in principle allow to estimate a men’s age from a male-female DNA mixture. When successful, this for instance would allow from a male-female mixed stain to differentiate between male relatives such as father and son in cases where autosomal STR profiling is not informative and a Y-STR haplotype match is observed. Here, we investigated the age predictive value of 270 male-specific Y-chromosomal CpG sites included in the Illumina Methylation Beadchip 450K microarray that passed our strict quality control. For this, we obtained 450K data from blood of hundreds of males with a wide age range (15–80 years old) using public databases, and normal- ized them using well-chosen procedures (ENmix and sva R packages). We discarded Y-CpGs with inter-quantile range (IQR) <0.1, and subsequently implemented elastic nets to successfully identify a subset of age-informative Y-CpGs. Using a finite set of Y-CpGs (n<30), we achieved a mean absolute deviation between true and predicted age of ~7 years in an independent dataset. Test- ing in other datasets and investigation of forensically relevant tissue types other than blood are currently ongoing. To our knowledge, this is the first study applying male-specific DNA methylation patterns for an age estimation strategy to investigate mixed male-female forensic samples.

666 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS NEW POLYMORPHISMS OF FORENSIC INTEREST

P584 - 38 INSERTION-DELETION (INDEL) MARKERS ANALYSIS AND ITS APPLICATION IN FORENSIC CASES Heitor Simoes Dutra Correa1, Venusia Cortellini1, Gloria Brescia1, Nicoletta Cerri1, Andrea Verzeletti1

1 University of Brescia, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Institute of Forensic Medicine, Brescia, Italy

The typing of short tandem repeats (STRs) is a common method used in forensic laboratories based on its good performance. However, because of the relatively large size of amplicons (150– 500 bp), most of them are unsuitable for degraded DNA and low copy number samples. So, there is a need to find new genetic makers with smaller amplicon size. Recently, some forensic researchers focused their attention to alternative and supplementary genetic markers in the human genome: insertion-deletion polymorphisms (InDels). They are diallelic, of smaller size, widely distributed throughout the genome and with a lower mutation rate compared to STRs. Due to their characteristics, InDels can aid forensic analysis even when a very small amount of DNA is available in a sample. In this study we tested the ability of a set of 38 InDel markers on genotyping different forensic samples containing low DNA concentration, already typed using the Identifiler kit but obtaining poor results. Statistical analysis was performed using the STRs results with appropriated softwares. After, InDel typing results were added to the STRs statistic calculations. The purpose of this work was to verify the actual usefulness of InDels analysis in order to improve the weight of the statistical analysis of challenging samples.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 667 P585 - A COMMON INDEL POLYMORPHISM OF THE DESMOGLEIN-2 (DSG2) IS ASSOCIATED WITH SUDDEN CARDIAC DEATH IN CHINESE POPULATIONS Yuzhen Gao1, Yan He2, Lijuan Li1

1 Soochow University, Dept. of Forensic Medicine, Suzhou, China 2 Soochow University, Dept. of Epidemiology, Suzhou, China

Sudden cardiac death (SCD) is referred to as sudden and unexpected death caused by cardiovascular diseases, in which a person preexisted heart disease or not. Compelling evidence indicates that SCD etiology have been predominantly affected by host genetic factors. However, how genetic variants play roles in the inherited risk component of SCD are still largely unknown. It has been reported that Desmoglein-2 (DSG2) mutations might be related to sudden death. In the present study, we used a candidate gene approach to investigate the associations between rs397729601 (a 2-base pair indel polymorphism) mapping to the 3 UTR of DSG2 with the risk of SCD. It is shown by logistic regression analysis that the risk of SCD has been significantly increased by the deletion allele of rs397729601 [odds ratio (OR) =1.51;′ 95 % confidence interval (CI) =1.12–2.05; P=0.00559]. Additional genotype-phenotype analysis was performed to evaluate the mRNA level, revealing that human myocardium tissues with the deletion allele showed higher expression of DSG2. Dual luciferase activity analysis was conducted in an in vitro reporter gene system, suggesting that DSG2 expression could be regulated by rs397729601 which interrupted the binding of miR-933–3p with DSG2. We concluded that rs397729601 may affect the expression of DSG2 through miR-933–3p regulation, contributing to SCD susceptibility. Thus, rs397729601 may be used as a potential marker for molecular diagnosis and genetic counseling of SCD. Our findings need to be validated through replication and further functional studies.

668 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P586 - A NEW APPROACH TO DETECT A SET OF SNP‑SNP MARKERS: COMBINING ARMS-PCR WITH SNAPSHOT TECHNOLOGY Ranran Zhang1, Yu Tan1, Hui Jian1, Yuqing Liu1, Shengqiu Qu1, Huan Tian1, Li Wang2, Meili Lv3, Weibo Liang1, Lin Zhang1, Yongqing Wang4

Microhaplotype[1] has become a new promising forensic genetic marker. Without the interference of stutter and high mutation rate as short tandem repeats (STR) and low polymorphism as single SNP, the microhaplotype composed of two SNPs, SNP-SNP, indicates strong application potential because of the shortest fragment and good polymorphism. Currently, the most common method to detect microhaplotypes is massively parallel sequencing (MPS), however the requirements of costs and instruments cumber its widely use in all the forensic laboratories. In this study, we screened out 23 new SNP-SNP loci and established a new detection method by associating multiplex ARMS-PCR and SNaPshot technology for forensic application. Firstly, we introduced additional deliberate mismatch at the antepenultimate base from the 3 end of primers for ARMS-based PCR for SNP1. Then, SBE primers for SNaPshot assay were designed as 20–25bp upstream complementary sequence next to the position of SNP2. Finally, 17 loci′ were built into two panels containing eight and nine loci respectively based on different SBE primer lengths and fluorescence colors. Another 6 loci were excluded because there were no appropriate ARMS primers. In brief, by combing ARMS and SNaPshot technology, it is easy and fast to profile the SNP1 and SNP2 orderly of the SNP-SNP haplotype based on CE platform. Our results suggested that the 17 loci have relatively high polymorphism as well as robust performance. Furthermore, this approach and SNP-SNP panel could offer a valuable complementary tool in detecting microhaplotypes. 1. K.K. Kidd, A.J. Pakstis, W.C. Speed, R. Lagace, J. Chang, S. Wootton, N. Ihuegbu: Microhaplotype loci are a powerful new type of forensic marker. Forensic Sci. Int. Genet. Suppl. Ser, 2013. 4(1): p. e123-e124.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 669 P587 - A STUDY OF QATARI POPULATION USING FORENSEQ™ DNA KIT Eida Almohammed1, Hadi Sibte2

1 Ministry of Interior Qatar, Qatar Forensic Laboratory, Doha, Qatar 2 University of Central Lancashire, School of Forensic & Applied Sciences, Preston, United Kingdom

Recent advances in Massively Parallel Sequencing (MPS) provide advantages over previously used technologies to analyse DNA samples in terms of speed and number of DNA markers that can be analysed simultaneously. The aim of this study was to evaluate the Illumina ForenSeq DNA signature kit and MiSeq FGx platform. The beta-version of the kit was used to genotype 150 Qatari samples that had been profiled using GlobalFiler™ kit on the ABI 3500. In this study, 150 Qatari population samples previously typed using the GlobalFiler™ kit were sequenced using the ForenSeq™ kit. The MPS workflow included the use of 2800M positive and negative controls to check the quality and consistency of the runs. A comparison of UAS results for autosomal STRs to GlobalFiler™ kit was done. In addition, tertiary analysis was done using STRait Razor software. This is the first study for the Qatari population using MPS methods and the results would be subsequently useful for casework purposes in the Qatar forensic laboratories.

670 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P588 - ANALYSIS OF 55 KIDD ANCESTRY SNP OF QATARI POPULATION COMPARED TO 139 WORLD WIDE POPULATION Eida Almohammed1, Hadi Sibte2

1 Ministry of Interior of Qatar, Qatar Forensic Laboratory, Doha, Qatar 2 University of Central Lancashire, School of Forensics and Applied Sciences, Preston, United Kingdom

SNPs are good predictors of ethnicity and serval panels have been published (1). The ForenSeq Signature kit (Illumina) offers coverage of 230 different markers including 55 ancestry SNPs (AISNPS). The ForenSeq Universal Analysis Software (UAS) provides the capability to analyse the sequencing data, visualise results and perform statistical estimates of biogeographic ancestry. The ancestry prediction capabilities in UAS are based on Principal Component Analysis (PCA) built on several reference populations included in the 1000 Genomes project. This set does not include Qatari population. Therefore, the ancestry prediction capabilities of the ForenSeq kit through sequencing on the MiSeq FGx were evaluated by profiling 150 Qatari population samples. The samples were collected from native Qatari population from different regions in Qatar. The data was analysed using STRUCTURE software. These data serves as an addition to the existing Middle Eastern population data for the 55 AISNPS. The results of this study are presented herein.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 671 P589 - APPLICATION OF A SNP-STR MULTIPLEX ASSAY FOR FORENSIC DNA MIXTURE INTERPRETATION Hui Jian1, Li Wang2, Yu Tan1, Ranran Zhang1, Yuqing Liu Liu1, Weibo Liang1, Lin Zhang1

Forensic mixtures often exist and play an important role in various forensic casework. Currently, a variety of molecular markers, including Y-STR, DIP-STR and SNP-STR have been widely used to address this problem1. SNP-STR is a compound genetic marker composed of a SNP locus and a closely linked STR. In conjunction with a PCR technique based on amplification refractory mutation system (ARMS), it can be used to resolve extremely unbalanced two-person DNA mix- tures2. In addition, this marker combines the advantages of both SNP and STR, thus providing modestly higher levels of polymorphism and possessing great potential in analysing the balanced two-person mixtures. 18 SNP-STR loci and three SNP-STR multiplex PCR panels were established and population study was carried out in a southwest Chinese Han population in our previous study. In this study, we firstly applied the multiplexes in a sexual-assault case to find out advantages and limitations in balanced two-person mixtures analysis. Results showed that likelihood ratios assigned to measure the strength of SNP-STRs’ DNA evidence were higher than autosomal STRs’. In conclusion, the SNP-STRs can provide significant value to the analysis of balanced two-person DNA mixtures. 1. Gill P., Brenner C. H., Buckleton J. S., et al., DNA commission of the International Society of Fo- rensic Genetics: Recommendations on the interpretation of mixtures(2006), Forensic science international. 160: 90–101. 2. Tan Y., Bai P., Wang L., et al., Two-person DNA mixture interpretation based on a novel set of SNP-STR markers(2018), Forensic Sci Int Genet. 37: 37–45.

672 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P590 - ASSOCIATING DONOR AND CELL TYPE USING RNA SNPs Margreet Van Den Berge1, Elske Dwars1, Rachel van Leeuwen1, Lydia Stravers1, Titia Sijen1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and organ tissues. Although RNA profiling is generally performed simultaneously and alongside DNA profiling from the same trace of evidence, DNA and RNA results are not readily linked e.g. due to expression differences intrinsic to mRNA analysis. Also, one contributor can donate more than one cell type and a single cell type can be given by multiple contributors. Therefore, an approach is desired that associates DNA and RNA profiling results enabling combined interpretation and reporting. Use of the sequence of the RNA molecules may be a solution as this can provide donor-specific information when single nucleotide polymorphisms (SNPs) in transcribed DNA regions are carried over to the RNA molecules. In this study, those regions were examined for SNP locations in various databases. Massively parallel sequencing (MPS) assays were designed to associate donor (DNA) and cell types (RNA), initially focussing on semen, blood and saliva as for these body fluids donor association may be prudent (multiple perpetrator rapes, violent cases with blood-blood mixtures, sexual assaults involving licking/kissing). Reference SNP profiles were obtained using DNA extracts, while trace profiles were obtained from RNA extracts derived from the corresponding body fluid thereby confirming cell type-specificity of the examined markers. Manual comparison of these DNA and RNA SNP profiles can be used to determine whether a person of interest may have contributed to the trace. Preliminary results show high potential for application in casework, especially when likelihood ratio calculations are performed to determine the strength of the weight of evidence.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 673 P591 - CONFIRMATION OF PHASED-INFERRED SNP HAPLOTYPE DATA OF 74 MICROHAPLOTYPE LOCI AND ANCESTRY PREDICTION BY MASSIVELY PARALLEL SEQUENCING Fabio Oldoni1, Leena Yoon1, Aishwaryaa Subramanian1, Sathya Prakash Harihar1, Sharon Wootton2, Robert Lagacé2, Ryo Hasegawa2, Joseph Chang2, Kenneth Kidd3, Daniele Podini1

Microhaplotypes (MHs) are multi-SNP loci within 300bp displaying multiple allelic combina- tions1. Within a locus all alleles have same size, low mutation rate and display no stutter. These features make them useful for human identification, ancestry inference, and mixture decon- volution2. Sanger sequencing/TaqMan® assay do not allow determining the cis/trans relationship among individual SNPs inferred using PHASE software3 while massively parallel sequencing (MPS) enables distinguishing the parental haplotypes by clonally sequencing of each DNA strand. This project confirmed by sequencing phased-inferred SNP haplotypes of 74 MHs loci and evaluated their ancestry prediction capabilities4, 5. A global set of >800 samples from Africa (Sandawe, Hausa), Europe (Danes, Khanty), South Cen- tral Asia (Laotians, Keralites), East Asia (Koreans, Atayal), Oceania (Papua New Guineans), Native America (Mexican Pimas), and America (Africans, Europeans, East Asians and Southwest Hispan- ics) was genotyped using an MPS 74 MH locus-assay totalling 230 SNPs on Ion S5™ (Thermo Fisher Scientific) platform. Overall >94 % concordance was observed between phased-inferred and MPS-determined haplotypes. However, differences were identified in haplotypes with multiple heterozygous SNPs due to missing/mistyped SNPs using TaqMan®or incorrect phase estimation. Ancestry prediction was evaluated by STRUCTURE analysis, which enabled the clustering of all samples within the main global population groups and correct ancestry prediction of unknown tested samples. These results indicate that computational/statistical phasing is a valuable/inexpensive approach for MH loci search6 and should be confirmed by sequencing. This 74-locus panel is an effective tool for complementing existing ancestry-biomarker assays.

674 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P592 - DEVELOPMENT OF A MULTIALLELIC INDEL FORENSIC ASSAY: INSERTION-DELETION SHORT MICROHAPLOTYPES Manuel Fondevila Alvarez1, Veronica Martos-Gago1, Jorge Amigo2, Christopher Phillips1, María Victoria Lareu Huidobro1

1 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic genetics unit, Santiago de Compostela, Spain 2 University of Santiago de Compostela, Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela, Spain

Development of simple biallelic markers for forensic purposes has been an important area of research activity in the last decade. Despite highly desirable characteristics, particularly their resistance to DNA degradation and PCR inhibition based on simple, very short amplicon sizes, several limitations of SNPs and InDels have been reported. One important drawback is the presence of just two alleles in most loci, which limits their individual statistical power for relationship testing or ability to deal with DNA mixtures. To improve the power of SNPs and Indels for forensic use, we have discovered and selected SNPs and InDels with multiple alleles. Here we describe the strategy adopted for finding InDels with improved forensic power. We selected sets of InDels grouped in pairs or trios in tightly-spaced groups located in genome lengths shorter than 120 basepairs. Each of these InDel groups effectively forms a microhaplotipe that can be genotyped by amplification of a single sequence fragment. To facilitate simple capillary electrophoresis-based detection, each microhaplotype InDel group was selected such that any combination of their respective alleles results in a unique amplicon length. Restricting the amplified fragments to short genome regions helped to maintain the advantage of short amplification, while improving the discrimination power of the individual loci selected. We selected sets of InDels forming short microhaplotype with unique haplotype lengths, readily applicable to human identification and relationship testing. A multiplex PCR assay for the amplification of a number of the selected markers has been optimized and trialed with an range of population samples to gauge variability and evaluate assay performance in routine capillary electrophoresis genotyping.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 675 P593 - ESTABLISHING MASS SPECTROMETRY-BASED IDENTIFICATION OF BODY FLUIDS FOR CASEWORK Stephan Kuhlmann1, Sascha Roocke2, Katalin Barkovits2, Jennifer Stepien2, Annette Dorn3, Katrin Marcus2

1 Polizei NRW, Landeskriminalamt LKA NRW, Düsseldorf, Germany 2 Ruhr-Universität-Bochum, Medizinisches Proteom Center, Bochum, Germany 3 Polizei Bayern, Landeskriminalamt BLKA, München, Germany

The identification of human body fluids in crime scene samples is an important task usually residing in the departments and institutions dealing with forensic genetics. Body fluid identification (BFI) complements the results of forensic DNA-analysis as it often allows conclusions about the relevance of samples according to the alleged crime. Well-established methods of BFI are mostly based on the indirect detection of certain marker-proteins e.g. via antibody-binding or enzymatic activity. Not all of these methods are confirmative in nature and some can be difficult to combine. Therefore a lot of effort has been made in recent years to develop new methods using genetic polymorphisms like mRNA or DNA-methylation patterns. A road less travelled however is the improvement of protein identification by using state-of-the-art mass-spectrometry. Benefits of this approach include: confirmative, human-specific results by directly detecting marker-peptides; not consuming the parts of the samples needed for DNA-analysis; and the capacity of multiplexing. Here we present the development and implementation of a mass-spectrometry based method for BFI at two police departments of forensic science in Northrhine-Westphalia and Bavaria (LKA NRW and BLKA), Germany, together with the Medical Proteome-Center (MPC) of University Bo- chum (RUB). On the peptide-level we have validated old and new markers for the identification of semen, saliva, blood and vaginal secretion, which can be detected alone and in mixtures with high sensitivity and specificity. Pros and cons of the workflow compared with the established routine are discussed. This work was supported by the European Commission (Innerer Sicherheitsfonds ISF).

676 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P594 - EURASIAPLEX II: A NOVEL SELECTION OF SNPs FOR REFINED DIFFERENTIATION OF EURASIAN ANCESTRIES Christopher Phillips1, Marika Porto1, Manuel Fondevila Alvarez1, María Victoria Lareu Huidobro1

1 University of Santiago de Compostela, Institute of Forensic Sciences, Forensic genetics unit, Santiago de Compostela, Spain

Although biogeographic ancestry prediction is now a well-established forensic test, we are yet to reach the limit of population group resolution possible with compact panels of Ancestry Informative Markers (AIMs). The degree of differentiation, and consequently, the predictive values are reduced when the test populations are closely related or admixed. In such cases, it is important to achieve high enough predictive values that allow a suitably reliable prediction of geographic origin. To obtain this, AIMs whose variability is informative specifically for the population ancestries to test are required. In previous work, we took the approach of step-wise application of several AIM-SNP panels that were complementary, in terms of their targeted population groups, to a general purpose conti- nentally-informative core set. We have enhanced the predictive performance of the original Eur- asiaplex panel; which was focused on differentiation of South Asian populations from European, East Asian and Middle East population groups. This new panel compiles AIM-SNPs that are complementary to both the original Eurasian population-informative panel and the 34-SNP core set. SNP selection includes bi-alleic, tri-allellic and tetra-allelic loci and these have been optimized for genotyping in a single-tube multiplexed PCR and Single Base Extension reaction using Ther- mo Fisher Scientific SNaPshot technology and capillary electrophoresis with standard protocols. The final optimized assay has been applied to a selection of Eurasian samples from different populations in order to generate allele frequencies that extend existing training sets for use in Snipper for the prediction of geographical origin of individuals from populations within Eurasia.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 677 P595 - EVALUATION OF A COSTUMIZED PANEL OF 117 NOVEL MICROHAPLOTYPES SELECTED FOR FORENSIC IDENTIFICATION IN TWO MPS PLATFORMS María de la Puente1,2, Christopher Phillips1, Catarina Xavier2, Jorge Amigo3, Ángel Carracedo1,3, Walther Parson2,4, María Victoria Lareu1

1 Forensic Genetics Unit - Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain 2 Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck, Austria 3 Fundación Pública Galega de Medicina Xenómica FPGMX, Santiago de Compostela, Santiago de Compostela, Spain 4 Forensic Science Program, The Pennsylvania State University, Pennsylvania, USA

Novel forensic microhaplotype (MH) candidates were compiled by querying 1000 Genomes Phase III data for segments of less than 120 bp with two or more polymorphic SNPs (minor frequencies ≥ 0.1). From systematic searches of candidate loci followed by detailed in-silico sequence quality screens, a total of 107 autosomal and 10 X-chromosome highly polymorphic MHs were selected to ensure a minimum distance of 10 Mb between syntenic markers for autosomes, or 5 Mb for the X-chromosome. The 117 MHs were combined into a single pool custom Ampliseq panel targeting FFPE-level DNA input (i.e. amplicon lengths of 125–175 bp). Libraries for two different MPS platforms -MiS- eq FGx and Ion S5- were prepared for a set of samples forming a simple validation framework. Validation DNAs comprised: (i) control and Coriell DNAs, to allow comparison with publically available data; (ii) dilution series of control DNAs; and (iii) artificially degraded DNA samples. Additional Ion S5 analyses were made of a series of artificial DNA mixtures at 1:1, 1:3, 1:7 and 1:15 ratios. Sequencing results were evaluated for quality parameters of coverage; strand bias; base misincorporation rates; and allele balance. Despite applying strict prior sequence quality checks, ~15 % of MHs showed discordant results, low coverage or alignment issues. Nevertheless, forensic sensitivity assessments showed promising performance, with profiles of a minimum 95 % completeness with 0.032 ng input DNA and degraded DNA (that had negative STR profiles). DNA mixtures were easily detected, but a wider range of contributors and ratios should be tested to investigate to what extent such forensic samples can be deconvoluted. This research was supported by grants ED481B 2017/088 (Xunta de Galicia) and BIO2016-78525 (AEI, Spain).

678 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P596 - EVALUATION OF ALU INSERTION POSITIONS OBSERVED IN SILICO IN JAPANESE WHOLE GENOME SEQUENCINGS Toshimichi Yamamoto1, Kaito Sano1, Ji yeon Lee1, Shiori Sato1, Takashi Yoshimoto1, Akira Ishii1, Yosuke Kawai2, Katsushi Tokunaga2

1 Nagoya University, Department of Legal Medicine and Bioethics, Nagoya, Japan 2 The University of Tokyo, Department of Human Genetics, Tokyo, Japan

Alu elements are the most abundant transposable interspersed repeats which exist more than one million copies in the human genome. We obtained about 5000 Alu insertion positions and the subfamily types in silico from the data of whole genome sequencings (NGS) in about 400 Japanese. To evaluate whether Alu elements are inserted at these positions in vitro actually, in order from the most allele frequency for the insertion, we selected totally the 80 positions for 40 AluYa5 and AluYb8 subfamilies, respectively. Then we designed each primer set for each position in the 200 bp of flanking sequences from UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly data. We could design for 37 and 38 positions of AluYa5 and AluYb8 subfamilies, respectively. How- ever, those for the rest 5 positions could not be designed due to no specific sequences in those flanking regions. We tried to amplify with 8 Japanese DNA samples using those 75 primer sets, and checked the insertions by agarose gel electrophoresis. Unfortunately, at the 2 positions of AluYa5 and 3 of AluYb8, only a band without insertion was observed for all the samples. For these 5 positions, we would have to confirm additionally by increasing the number of DNA samples because of different allele frequencies from those of NGS. At the other 70 positions, the bands with the expected sizes inserted were observed. Alternatively, some allele frequency data were significantly different from NGS data. The different subfamily from in silico was also obtained by Sanger sequencing although only one position was analyzed. Accordingly, it was suggested that the Alu insertion position data in silico are almost reliable although the frequencies and subfamily might be different from data in silico at some of the positions.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 679 P597 - EVALUATION OF MICROHAPLOTYPES – A PROMISING NEW TYPE OF FORENSIC MARKER Adam Staadig1,2, Andreas Tillmar1,2

The forensic field is in a continuous need of improvements due to the various and complex issues in forensic investigations, in addition with its intricate sample material and quality. Stan- dard short tandem repeat (STR) analysis has limitations when working with highly degraded samples due to their relatively long fragment length. Furthermore, mixture samples i.e. DNA samples origin from two or more individuals, can be difficult to separate with STR analysis due to stutter artefacts caused during amplification. The development of MPS technology has enabled the discovery of several new types of forensic markers where microhaplotypes (MHs) are one of these promising novel genetic markers. MHs are commonly less than 300 nucleotides in length and consist of two or more closely linked SNPs. The short distance of the SNPs implies a low recombination rate and each MH can be sequenced within a single sequencing read, resulting in the haplotype of that particular region. These advantages makes MHs convenient for degraded samples, also, the stutter problem is eliminated making it a suitable marker of choice for mixture analysis and due to the low recombination rate it suits well for ancestral predictions and human identifications. We will present results from 72 samples analyzed with QIAseq MicroHaplotype panel (Qiagen). Library preparations were done with the GeneRead protocol (Qiagen), followed by sequencing on a MiSeq FGx (Illumina). We performed sensitivity studies and could detect alleles at concentrations as low as 100 pg. We also studied mixture samples with two contributors for which alleles were detectable down to the level of 1:100. To conclude, the results of this initial study are promising for further implementation of microhaplotypes as a new forensic marker.

680 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P598 - EVALUATION OF TWO DNA/RNA CO-EXTRACTION METHODS FOR BODY FLUID IDENTIFICATION IN FORENSICS Sara Loureiro1, António Amorim2, Laura Cainé3, Benedita Silva4, Iva Gomes5

1 Forensic Sciences and Medical Education- Faculty of Medicine FMUP- University of Porto- National Institute of Legal Medicine and Forensic Sciences- Porto- Portugal, Department of Public Health, Porto, Portugal 2 Institute for Research and Innovation in Health i3S- Institute of Molecular Pathology and Immunology of the University of Porto IPATIMUP- Department of Biology- Faculty of Sciences FCUP- University of Porto, Population Genetics and Evolution, Porto, Portugal 3 National Institute of Legal Medicine and Forensic Sciences- Porto- Department of Public Health- Forensic Sciences and Medical Education- Faculty of Medicine FMUP- University of Porto- School of Law- University of Minho- Braga- Portugal, Genetic and Forensic Biology, Porto, Portugal 4 National Institute of Legal Medicine and Forensic Sciences- Porto- Portugal, Genetic and Forensic Biology, Porto, Portugal 5 Institute for Research and Innovation in Health i3S- Institute of Molecular Pathology and Immunology of the University of Porto IPATIMUP- Porto- Portugal., Population Genetics and Evolution, Porto, Portugal

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work two methods, for DNA and RNA co-extraction, were tested and compared.The kit ExtractME RNA & DNA Kit (Extractme, BLIRT S.A., Poland) and a quick, simple manual based protocol, adapted from Shojaie N. et al. [Protocol Exchange (2014), doi:10.1038/protex.2014.036], were used to simultaneously extract both types of nucleic acids from semen samples. As a first quality control (QC) check, DNA and RNA quantifications, as well as purity assessment ratios (260/230 and 260/280), were estimated by Nanodrop 1000 Spec- trophotometer (Thermo Fisher Scientific, USA). High quantities of both DNA and RNA were obtained by both methods allowing for the use of extracts in downstream applications such as endpoint PCR. When comparing the ratios of QC obtained by spectrophotometry readings, the commercial kit provided better results overall. Results suggest that the manual method allowed for more carryover of contaminants absorbing at 230nm and 280nm as the purity ratios were below the accepted ranges. Nevertheless, these ratios can be further improved by purification of both DNA and RNA extracts. Finally, to assess the use of the tested protocols for body fluid identification, the gene expression profiles of two semen mRNA specific markers were compared among methods.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 681 P599 - FORENSIC EVALUATION OF SE33 MARKER IN INDIAN POPULATION USING GLOBAL FILER, FUSION 6C AND PANGLOBAL AUTOSOMAL STR MULTIPLEX Pankaj Shrivastava1, Kamlesh Kaitholia1, Shivani Dixit2, Hirak Ranjan Dash1

1 State Forensic Science Laboratory- Sagar MP India, DNA Fingerprinting Unit, SAGAR, India 2 State Forensic Science Laboratory-Sagar MP India, DNA Fingerprinting Unit, Sagar, India

Capillary electrophoresis based forensic DNA typing using PCR with multiplex STR kit gold standard now and is used for identification worldwide. But even one mismatch in the studied markers results in an inconclusive report. This deficiency is suggested to be taken care of by using alternative markers and/or by increasing the number of markers. Three new generation multiplex autosomal STR systems namely Global Filer (Thermo), PowerPlex Fusion 6 C (Prome- ga) and Panglobal (Sure ID) incorporating new 20 CODIS and the SE 33 marker were used on 819 samples to observe the forensic performance of SE33 marker. The study presents the first forensic evaluation of SE33 marker included in new generation multiplex systems being used for forensic DNA typing. A total of 52 alleles were observed on SE 33 marker in the studied population. Forensic parameters including allele frequency, Power of discrimination, Polymorphism information content, Power of exclusion, Paternity index, Observed and Expected Heterozy- gosity, Matching Probability, Shannon’s Information Index and Fixation Index are reported for the studied marker. Forensic application and trio analysis will also be highlighted in this study using the three multiplex kits.

682 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P600 - GENOME-WIDE CNV ANALYSIS IN THE CZECH POPULATION AND A NEW CONCEPT FOR DISTANT RELATEDNESS DETERMINATION Marie Korabecna1, Alzbeta Zinkova1, Iva Brynychova1, Halina Simkova2, Jan Geryk3

1 First Faculty of Medicine Charles University, Institute of Biology and Medical Genetics, Prague, Czech Republic 2 First Faculty of Medicine Charles University, Institute of Biology and Medical Genetics, Prague, Czech Republic 3 Second Faculty of Medicine Charles University, Department of Biology and Medical Genetics, Prague, Czech Republic

Introduction: Population studies demonstrated that all individuals carry multiple Copy Number Variants (CNV) of 1kb in size or greater in their genome. Due to low population frequency of many such benign variants, we decided to explore a new concept - application of CNV detection for distant relatedness determination. Interpretation of results is based on correct evaluation of the extent of each detected variant. We demonstrate the proof of principle and population data based on the whole genome sequencing (WGS) of 100 healthy individuals. Materials and Methods: We analysed a large four-generation pedigree to follow the segregation of CNVs and reproducibility of their calls using Agilent Sure Print G3 Human Microarray 2x 400K. Population data were based on WGS of 100 individuals generated using NovaSeq 6000 (Illumi- na). Results: We analysed a large four-generation pedigree using microarrays. We followed three times the segregation from a common ancestor to the fourth generation and four times to the third generation. Typically we detected around 30 CNVs per individual using microarray and we followed the segregation with high precision (identical genomic coordinates for the analysed CNV in different individuals). Then we demonstrated the potential of WGS with regard to the massively increased number of detected CNVs per individual together with population study of bialellic CNVs in the Czech population. Conclusions: We provided the evidence that CNV detection based on WGS and appropriate population data may represent a handy tool for distant relatedness determination. Acknowledgments: Supported by the Ministry of Interior of the Czech Republic grant no.VI20172020102.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 683 P601 - GENOME-WIDE SEARCH OF DIP-STRs CANDIDATES BY USING THE 1000 GENOME PROJECT DATASET Diana Hall1, Loic Baerlocher2, Vincent Castella1

1 Unité de Génétique Forensique- Centre universitaire romand de médecine légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, Switzerland 2 Fasteris SA, Fasteris SA, Plan-les-Ouates, Switzerland

DIP-STRs are compound markers formed by a deletion/insertion polymorphism (DIP) linked to a Short Tandem Repeat (STR). They enable the deconvolution of unbalanced DNA mixtures from two individuals, up to 1,000-fold excess of major contributor. A forensic set developed according to forensic standards was validated in casework. Yet, to promote the widespread use of this original approach among forensic practitioners, more markers need to be developed. Here, we describe the first genome-wide list of DIP-STR candidates identified by using the most comprehensive whole genome sequencing dataset: the 1000 Genomes project. Using the Phase 3 release comprising annotated genotypes for 2,504 individuals from 26 different populations, we found 61,877 DIPs with a sequence variant of at least three bases and minor allele frequency of 0.2 and 31,416 polymorphic STRs with the most frequent allele less than 0.8. DIP and STR spanning less than 200 bp are 474 and half of them span less than 100 bp. These markers are all located in non-coding regions. Finally, we estimated DIP-STR haplotype counts for the 474 candidates by phasing individuals who are homozygous for the DIP or for the STR. Based on these data, we selected 30 novel DIP- STRs with optimal features such as: mean heterozygosity of 0.81, ranging from 0.71 and 0.91, mean number of alleles equal to 18, ranging from 7 to 27 and an average size of 143 bp, ranging from 46 bp to 236 bp. In conclusion, these data show that several hundreds of DIP-STR candidates exist throughout the genome. Further development of an efficient DIP-STR panel comprising about 30 markers is feasible, and will be soon available.

684 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P602 - INVESTIGATION ON RAPIDLY MUTATING Y-STRS MULTIPLEX IN INDIAN POPULATION: A PILOT STUDY Pankaj Shrivastava1, Rashed Alghafri2, Almheiri Reem3, Mishra Aditi1,4, Ramkishan Kumawat5,6, Shivani Dixit1,6, Divya Shrivastava6

1 State Forensic Science Laboratory- Sagar MP India, DNA Fingerprinting Unit, Sagar, India 2 Dubai Police General Head Quarters- Dubai- United Arab Emirates, General Department of Forensic Sciences and Criminology, Dubai, United Arab Emirates 3 United Arab Emirates University, Biology Department, Al-Ain, United Arab Emirates 4 Raksha Shakti University, Department of Forensic Science, Ahmedabad, India 5 State Forensic Science Laboratory- Jaipur -Rajasthan, DNA Division, Jaipur, India 6 Jaipur National University, Biotechnology, Jaipur, India

Importance of rapidly mutating (RM) Y- STRs) is already established and is evident with the addition of seven RM Y-STR markers in new generation Yfiler Plus (YFP) multiplex system. Perfor- mance of the 7 RM Y-STRs included in YFP is evaluated in central Indian population. We also report here a pilot study on genetic polymorphism using 13 Rapidly Mutating Y STRs (RM Y-STR) on Indian population. Forensic parameters including allele frequencies, gene diversities, discrimination capacity and Haplotype diversity will be calculated for the amplification assays. Forensic application and kinship analysis will be highlighted in this study.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 685 P603 - MPS-BASED COMPARISON OF mtDNA HETEROPLASMY IN HAIRS AND CORRESPONDING BUCCALS TO INFORM MPS INTERPRETATION GUIDELINES Kristiaan van der Gaag1, Sophie Smit1, Stijn Desmyter2, Lourdes Prieto3, Titia Sijen1

1 Netherlands Forensic Institute, Division of Biological Traces, The Hague, Netherlands 2 Belgian Institute for Forensic Science and Criminology NICC, Laboratory for Genetic Identification, Brussels, Belgium 3 Universidade de Santiago de Compostela- Espana, Instituto de Ciencas Forenses, Santiago de Compostela, Spain

Heteroplasmic variation (HPL) can be a challenging factor for forensic interpretation of mitochondrial DNA (mtDNA) data, especially when individual hairs are involved. Somatic mutation or a genetic bottleneck in each hair follicle may cause HPL between hairs and other tissues which can lead to apparent homoplasmic mismatches between a buccal reference and a hair from the same individual. Since traditional Sanger sequencing cannot detect minor components present at less than ~20 % at a mixed position, the evidential strength of mtDNA analysis is impacted by the fact that a (single) mismatch cannot be excluded. The introduction of Massively Parallel Sequencing (MPS) not only expands the range of mixture detection to reveal more low level contributions, but also provides the opportunity to reveal linkage between mixed variants within the same fragment. In this study, we analysed 26 buccal reference samples and 475 hairs from the corresponding donors for the mtDNA Control Region using MPS using a 3 % variant calling threshold. For each donor, the frequency of HPL in the buccal sample and corresponding hairs was compared. Pre- viously, the samples had been analysed through Sanger and various mismatches occurred in the sense that a homoplastic variant in a hair was not detected as homo- or heteroplasmic variant in the corresponding buccal. With MPS analysis, all homoplasmic variants in hairs were observed in the buccals with (low-level) HPL contributions of at ≥7.5 %. From this information, considerations for interpretation of MPS mtDNA data in forensic casework were obtained. Moreover, we revisited the results from mtDNA analyses on hair samples that were analysed since we introduced MPS for mtDNA casework a year ago, to assess the impact of the interpretation guidelines.

686 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P604 - PERFORMANCE OF A MASSIVE PARALLEL SEQUENCING MICROHAPLOTYPES ASSAY ON DEGRADED DNA Chiara Turchi1, Filomena Melchionda1, Mauro Pesaresi1, Paolo Fattorini2, Adriano Tagliabracci1

1 Polytechnic University of Marche, Department of Biomedical Sciences and Public Health, Ancona, Italy 2 University of Trieste, Department of Medicine- Surgery and Health, Trieste, Italy

Massively parallel sequencing (MPS) has allowed to analyze a new type of forensic genetic marker, known as microhaplotypes (MHs). A MH locus is defined as “a short region of DNA characterized by the presence of two or more closely linked SNPs”. MHs appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Moreover MHs are potentially suitable for the analysis of degraded DNA samples. In a first pilot study, we evaluated the genetic variations of 87 MH loci by using MPS, in order to make inference about their usefulness in forensics. The resulting combined matching probability value was equal to 5.7 x 10-63. As the amplicons size of this explorative panel was too large to be used for degraded DNA typing, we selected a subset of 29 MH and designed a new panel, with amplicons sizes below 180 bp. The principal aims of this study were to validate the 29-MH panel and to investigate its effectiveness with low amounts of degraded samples. We genotyped a large set of real forensic samples together with a set of artificially degraded DNAs. The availability of a reference sample (“same donor” or “first degree relative”) was a recruitment criterion. Also, a sensitivity test was assessed by a set of 2800M DNA dilutions. MPS assays were performed by Ion PGM and Ion Gene Studio S5 System. This study investigated the concordance typing results observed in the sensitivity test and evaluated the performance of the 29-MH panel with respect to the DNA quantity and the DNA degradation index, in order to optimize the analytical conditions of such challenging samples. Finally, consideration about the bioinformatics analysis and the analytical threshold required to reliably call a microhaps allele were made.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 687 P605 - PROBABILISTIC GENOTYPING OF MICROHAPLOTYPES Daniele Podini1, Drew Bader2, Fabio Oldoni2, Charles Brenner3, Kenneth Kidd4

1 GWU, Forensic Sciences, WASHINGTON, USA 2 GW, Forensic Sciences, Washington, USA 3 DNA-View Mixture Solution, Research, Okland, USA 4 Yale, Genetics, New Haven, USA

Microhaplotypes (MHs) are an emerging forensic DNA marker characterized by multiple SNPs within a short distance of each other displaying several allelic combinations. Although less polymorphic than short tandem repeat polymorphisms (STRs) they are characterized by showing same-size alleles resulting in being immune to preferential amplification of shorter alleles within a locus. Furthermore, their analysis does not generate stutter artifacts and have a lower mutation rate than STRs making them a more effective markers for relationship testing. Several MH-multiplex panels have been reported in the past two years, some targeting over 70 loci. Casework implementation of such large panels is only feasible if paired with probabilistic genotyping (PG) as manual deconvolution of complex mixtures would be excessively time consuming and not compatible with conventional forensic DNA laboratory operations. To evaluate the feasibility and performance of PG of MHs LRmix Studio and DNA-View Mixture Solution were adapted to processing MH data from 74 loci; RFU values were replaced by allele sequence coverage. Over 30 different DNA mixtures were prepared ranging from two (2) to five (5) contributors in multiple donor-ratio combinations with 1 ng and 10 ng input DNA. As an example, a five-person mixture with donor ratio 10:5:2:1:1 (ABCDE) yielded an LR = 8.75E+3 with LRMix Studio and 1E+78 with DNA-View Mixture Solution when setting Hp = A + B + three unknowns and Hd = A + four unknowns. As expected PG based on fully continuous modeling of the data yielded a much higher LR given the capability of using allele coverage in the deconvolution of the mixtures. Results show that MHs data can be interpreted using PG making this novel forensic marker one step closer to casework implementation.

688 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P606 - PROPOSED A NEW NOMENCLATURE FOR MICROHAPLOTYPES Jing Zhu1, Shengqiu Qu1, Yinji Wang1, Lu Yin1, Duo Peng1, Bin Long2, Wang He2, Jiong Mao3, Hui Wang3, Weibo Liang1, Lin Zhang1, Dan Chen3

1 West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Department of Forensic Genetics, Chengdu 610041- Sichuan, China 2 Sichuan Police College, Department of Forensic Genetics, Luzhou 646000- Sichuan, China 3 Institute of Forensic Science- Chengdu Public Security Bureau, Department of Forensic Genetics, Chengdu 610081- Sichuan, China

Microhaplotype is an emerging forensic genetic marker. A unified and standardized nomenclature is required. Kenneth K. Kidd has proposed a nomenclature for microhaplotype marker[1]. The nomenclature involves a simple root consisting of “mh” followed by the chromosome number and the unique characters established by the authors. Professor Kidd suggests that the symbol of microhaplotype marker should be indication of the laboratory followed by characters unique to the chromosome and laboratory. Our team proposed a new nomenclature for microhaplotypes. The new one mainly involves three parts: 1. which chromosomes the loci are located on, 2. the order that we defined the loci, and 3. the version (A/B/C…) that defined by different groups of SNPs. For example, a locus that contains 6 SNPs (rs150238110, rs12358044, rs10905667, rs11256509, rs190776965 and rs181658358) is located on chromosome 10 and is defined as No.4. It is named as mh10zl4A (The letters “mh” and “zl” are used following the Kidd’s proposal). The allele frequencies of 26 populations are calculated based on 1000 Genomes Phase 3 data. The minor allele frequency (MAF) of rs150238110 was 0 and only allele G was present in several populations (e.g., CHB and CHS). The MAF of rs11256509 was 0 in population YRI. According to the new nomenclature, the locus should be named as mh10zl4B (rs12358044, rs10905667, rs11256509, rs190776965 and rs181658358) in population CHS and CHB, and mh- 10zl4C (rs150238110, rs12358044, rs10905667, rs190776965 and rs181658358) in population YRI. [1] K.K. Kidd, Proposed nomenclature for microhaplotypes, Hum Genomics 10(1) (2016) 16.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 689 P607 - SEQUENCE CHARACTERIZATION OF MICROVARIANT ALLELES AT DYS627 AND DYS458 Yiping Hou1, Min Lang1, Feng Song1, Yi Ye1, Hong Zhu1, Zheng Wang1

1 Sichuan University, Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Chengdu, China

Y chromosome short tandem repeats (Y-STRs) have been widely used in genetic applications and forensic casework. Recently, we found two intermediate alleles, the DYS627 allele 24.1 and the DYS458 allele 15.3 from Chinese Han population. The two microvariants have not been recorded by the YHRD database. We have examined the molecular structure of these allelic variants by Sanger sequencing. The results showed that this intermediate allele at DYS627 was confirmed as 24.1, the sequence of which showed a base “A” insertion in the 13th repeat unit, and the intermediate allele at DYS458 was confirmed as 15.3 caused by a base “G” deletion in the 12th repeat unit. This may be important for individual identification and paternal kinship testing. Besides, more microvariant alleles detected can be enriched in the Y-STR database.

690 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P608 - THE APPROPRIATENESS OF THE ANALYSIS OF SOME KNOWN micro-RNAs TO DETECT THE PRESENCE OF SEMEN IN OLD STAINS Massimo Lancia1, Simona Severini2, Federica Tommolini1, Eugenia Carnevali2

1 University of Perugia, Dep. Surgical and Biomedical Sciences - Section of Legal Medicine and Forensic Sciences, Perugia, Italy 2 Hospital of Terni, Section of Legal Medicine and Forensic Sciences, Terni, Italy

Recently miRNAs have gained notoriety for having revealed useful biomarkers. The advantage of using microRNAs as biomarkers lies in their ease of use and the accuracy with which they can be measured as well as their extreme tissue specificity. Furthermore, these small molecules are ex- ceptionally stable in laboratory treatments, unlike larger RNA molecules such as messenger RNA. In the forensic field, the use of microRNAs to confirm biological evidence has became a powerful tool of crucial importance for the reconstruction of a crime scene. Semen can be significant evidence in some types of crime, particularly sexual assault. However, a diagnosis of sperm is often difficult for several reasons, such as trace degradation and the presence of azoospermia, which are still present when using morphological, immunological and enzymatic methods. Re- cent studies show that the use of micro-RNAs overcomes both the problem of degradation (due to their high resistance to exogenous factors) and the absence of spermatozoa in azo- ospermic individuals. In this study, some semen-specific microRNAs (miR-10a-5p, miR-888–5p, miR891a-5p, miR135a-5p, miR135b-5p) on old semen stains to assess their suitability in forensic casework were tested. The expression of this set of markers was evaluated using a miRCURY LNA Universal RT microRNA PCR System. LNA technology showed interesting properties for molecular hybridization, including enhanced specificity in allele-specific PCR and can be used to overcome the difficulties of studying very short sequences. We concluded that the cellular specificity of the studied micro-RNA set makes it possible to identify the presence of semen in old stains with high specificity and consequently with high probative value in a court of justice.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 691 P609 - THE POTENTIAL FOR ANCESTRY INFERENCE OF DIP‑SNP Shengqiu Qu1, Jing Zhu1, Yinji Wang1, Meili Lv2, Lu Yin1, Li Wang3, Hui Jian1, Yu Tan1, Ranran Zhang1, Yuqing Liu1, Weibo Liang1, Lin Zhang1

1 Sichuan University, Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Chengdu, China 2 Sichuan University, Department of Immunology, West China School of Basic Medical Sciences and Forensic Medicine, Chengdu, China 3 Sichuan University, Department of Obstetrics and Gynecology, West China Second University Hospital, Chengdu, China

Since the introduction of microhaplotypes by Kidd in 2013, more and more studies on this kind of markers have been done. As different forensic applications require, more multi-variant sets are used as haplotype markers, such as microhaplotype, SNP-STR, DIP-STR, DIP-SNP. DIP- SNP combines the advantages of length polymorphism of insertion/deletion and sequence polymorphism of SNP, and both polymorphic markers have lower mutation rates than STR. Moreover, the widespread of INDELs and SNPs in the human genome makes DIP-SNPs easy to search. These all make DIP-SNP more advantageous for ancestry inference. In our study, we used the 1000 Genomes Project Phase 3 database to select 11 DIP-SNPs to form a set to evaluate the ability of DIP-SNPs for ancestry inference. The frequency and genotyping of each SNP and INDEL in different populations were obtained with VCFtools. And then, the frequency of each DIP-SNP in different populations was obtained using Haploview. Finally, we used the 11 DIP- SNPs set to analyze the population structure of different population with STRUCTURE and PCA. The results indicate that the DIP-SNP markers possess the potential of ancestry inference and can be used to further distinguish subgroups.

692 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P610 - THE STUDY OF 95 IDENTITY SNPs FOR QATARI POPULATION Eida Almohammed1, Sibte Hadi2

1 Ministry of Interior of Qatar, Qatar Forensic Laboratory, Doha, Qatar 2 University of Central Lancashire, School of Forensic and Applied Sciences, Preston, United Kingdom

A single nucleotide polymorphism (SNP) is a variant at a nucleotide position in the chromosome. In the forensic context, the major advantage of these markers is their possible usage with highly degraded DNA as in disaster victim identification and forensic samples. Approx- imately 100 SNPs have to be investigated to give a match probability equal to that obtained from 20 STRs (1). The Illumina ForenSeq kit covers most loci of forensic interest. There are numerous SNP panels which are designed as potential for identification of individuals and they include 52 SNP panel and 95 identity iSNPs which is included in Signature DNA kit. One hundred and fifty (150) reference samples sequenced using the ForenSeqTM DNA Signature kit. The results have clearly demonstrated the potential use of MPS methods to study the 95 iSNPs of Qatari population. This study serves as the first investigation into Qatari population genetics with respect to forensically-relevant loci as well as the first set of population data reported using the ForenSeq™ DNA Signature Prep kit. STR and identity SNP allele frequencies have been reported for 150 Qatari samples.The data presented in this study produce combined STR and identity SNP RMPs of the combined match probability of 2.9×10-68 and 1.12×10-75 for length- based and sequence-based autosomal STR alleles respectively. The magnitude of these RMP values highlights the power of a combined STR and SNP approach towards source attribution in forensic DNA typing. References: (1) GILL, P., et al. 2001. DNA Commission of the International Society of Forensic Genetics: recommendations on forensic analysis using Y-chromosome short tandem repeats. Legal Medicine, 3, 252–257.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 693 P611 - THE UNIQ-TYPER™ Y-10 GENOTYPING IN SOUTH AFRICAN POPULATIONS: NOVEL ALLELES, SEQUENCE VARIATION AND ALLELIC LADDER UPDATES Mohaimin Kasu1, Maria Eugenia D’Amato1

1 Forensic DNA Laboratory, University of the Western Cape, Biotechnology, Cape Town, South Africa

The Y-chromosome short tandem repeat (Y-STR) panel of the UniQ-Typer™ Y-10 was previously shown to improve male discrimination in South African native populations compared to the core Y-STRs of commercial kits. Population wide genotyping across 15 South African populations provided a series of novel alleles and sequence variants across its panel of Y-STRs namely: DYS710, DYS518, DYS385ab, DYS644, DYS612, DYS626, DYS504, DYS481, DYS447 and DYS449. In this report a total of 153 cloned and sequenced Y-STR alleles were incorporated into an allelic ladder for forensic applications. Using the cloning approach, a workflow is presented to assist a bulk ladder production process. From the genotyping and sequence analysis, we encounter a series of novel alleles at loci DYS385, DYS644, DS447, DYS710 and DYS504 and a total of 94 novel sequences. Repeat pattern variants encountered for 39 % of the novel sequences identified loci in the panel promising for the discrimination between isometric alleles. Furthermore, rare variant associations between loci were found restricted to certain populations. This was observed for individuals with haplotypes containing duplications at DYS710 and single base deletion variants at DYS644. The variation reported herein were mainly encountered in individuals of African paternal ancestry. This degree of variation we identified from a relatively small sample size encourages the need for large scale variant characterizations which may improve discrimination between male haplotypes.

694 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P612 - TRIAL TO SEARCH FOR MITOCHONDRIAL DNA MUTATION ASSOCIATED WITH CANCER DETECTED BY MASSIVE PARALLEL SEQUENCING Katsuya Honda1, Keishi Umino1, Shigeru Akanuma1, Yayoi Iwabuchi1, Hisanori Muramatsu1, Yukiko Sugano1, Fujio Ishizawa1

1 University of Tsukuba, Department of Legal medicine, Tsukuba city, Japan

We performed DNA analysis of blood and cancer-affected organs in patients with cancer and situs inversus, which is considered to be a pre-cancerous condition. The total mtDNA genome of patients was analyzed and compared with that of healthy Japanese people. Long-range PCR was performed with Nexterapreparation spanning the entire human mitochondrial genome (16,569 bp). Library sequencing on MiSeq (Illumina) was followed by data analysis with the mtD- NA variant analyzer. The analytical procedure was carried out in accordance with the instructions of “Human mtDNA Genome” ( Illumina). We found that some mtDNA sequences in cancer cases were unique and specific, being rarely found in the normal population. Additionally, the mtDNA from cancer and situs inversuscases was found to share the same mutations at some sites. Surprisingly, the findings suggested we can diagnose or predict cancer by surveying mitochondrial mutations of the blood. Recently, in clinical medicine, focus has been drawn to so-called liquid biopsies for the DNA analysis of blood. In this method, a focus is affected by mutation of the nuclear DNA. The analysis of mitochondrial DNA has mainly been studied in forensic medicine. Our results revealed its potential use also for cancer diagnosis.We already reported specific polymorphism and the collapse of maternally inherited mitochondrial DNA in sex chromosomal aberrations including Klinefelter’s syndrome1)and Turner’s syndrome2). The relationship between cancer and mitochondrial DNA polymorphism revealed here could open up a new research field. We suspect that certain mtD- NA polymorphisms are closely related to normal cells transforming into cancer ones via certain mechanism. 1. Honda K, et al: BBRC. 2002, 297: 341–345 2. Honda K, et al: FSIGSS. 2015, 5: e375–e377

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 695 P613 - VALIDATION OF THE CONSISTENCY OF cSNP ANALYSIS BETWEEN DNA AND RNA USING SNAPSHOT METHOD Yiping Hou1, Shouyu Wang1, Zheng Wang1, Ruiyang Tao1,2, Feng Song1

1 Sichuan University, Institute of Forensic Medicine, West China School of Basic Science & Forensic Medicine, Chengdu, China 2 Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai, 200063, China

The use of coding region single nucleotide polymorphisms (cSNPs) was recently proposed as a potential method for individual identification because it allowed mRNA profiling and DNA typing to be performed simultaneously. Nevertheless, availability of this approach still needs some further validation in different aspect. In this study, we have initially selected several SNP loci located in mRNA molecules that were confirmed to be highly expressed in certain body fluids. Both coding regions (CDRs) and untranslated regions (UTRs) were taken into consideration during the screening. Genomic DNA (gDNA) and total RNA from corresponding body fluid samples were isolated, followed by the synthesis of first-strand complementary DNA (cDNA) using purified RNA samples. Subsequently, the genotypes of these SNPs were respectively determined with gDNA and cDNA by using SNaPshot method. The PCR primers for cDNA were designed to span an intron in order to ensure that the amplification products were not due to the presence of potential DNA contamination. Our study revealed a high consistency of cSNP analysis between DNA and RNA on capillary electrophoresis platform, which highlighted the potential use of cSNP in forensic investigation.

696 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P614 - VARIANT ALLELE 6.2 AT LOCUS D19S433 IN SYRIAN FAMILY SAMPLES Sasitaran Iyavoo1, Thomas Haizel1

1 Anglia DNA Services, Scottow Enterprise Park, Norwich, United Kingdom

Anglia DNA Services uses AmpFℓSTR® Identifiler® PCR Amplification kit for paternity and relationship testing. This kit contains 15 STR loci including locus D19S433 which covers alleles 9–17.2. Variant allele 6.2 out of this marker range was found in the Syrian family samples submitted for an immigration case consisting a father, a mother and two children. Buccal swabs from all four individuals were processed and DNA profiles were developed. Variant alleles were assigned based on the amplicon size using the allelic ladder provided by the manufacturer. Extracted DNA samples were re-amplified and re-analyzed to confirm these variant alleles. Locus D19S433 was not studied in the Syrian population [1], thus this variant allele has not been reported previously. This variant allele was also found in another immigration case at Anglia DNA Services where the mother and the child were classified as Kuwaitis. Variant allele 6.2 was observed in the Iraqi Arab population data [2] and in individuals known to have Turkish origin [3]. These show this variant allele could be specific to the Middle Eastern populations. 1. L. Abdin, I. Shimada, B. Brinkmann, C. Hohoff, Analysis of 15 short tandem repeats reveals significant differences between the Arabian populations from Morocco and Syria, Legal Med. 5 (2003) S150–S155. 2. M.M. Farhan, S. Hadi, A. Iyengar, W. Goodwin, Population genetic data for 20 autosomal STR loci in an Iraqi Arab population: Application to the identification of human remains, Forensic Sci. Int.: Genet. 25 (2016) e10–e11. 3. M. Heinrich, H. Felske-Zech, B. Brinkmann, C. Hohoff, Characterisation of variant alleles in the STR systems D2S1338, D3S1358 and D19S433, Int. J. Legal Med. 119 (2005) 310–313.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 697 P615 - WHOLE miRNAome ANALYSIS IN SALIVA STAIN BASED ON MASSIVE PARALLEL SEQUENCING: A PILOT STUDY Chen Fang1, Huijuan Wu1, Jing Zhao2, Xu Liu1, Wenli Liu1, Jialin Qian1, Jiangwei Yan3

1 Beijing Center for Physical and Chemical analysis, Judicial Identification Center of Beijing Center for Physical and Chemical Analysis, Beijing, China 2 Chinese Academy of Sciences, Beijing Institute of Genomics, Beijing, China 3 Shanxi Medical University, School of Forensic Medicine, Taiyuan, China

MicroRNAs (miRNAs) are mainly applied in body fluid or tissue identification due to their cell type speciﬁc expression patterns and stability against degradation. Saliva, one of the most valuable sources of biological evidence, provides effective information during case investigations. However, the exploration of miRNAs in saliva, especially for aged and environmentally compromised forensic samples is currently lacking. Recent developments in massively parallel sequencing (MPS) technology provide the opportunity to establish a whole-genome miRNA profile with high throughput and efficiency, but the MPS analysis for miRNA profiles from saliva stain has not been reported yet. In this study, we provide a novel method to explore miRNA profiles based on MPS using saliva stains which stored for 2 months from 10 μL saliva. An average of 406 M sequencing data was produced and 72 known miRNAs and 33 novel miRNAs were detected. Among the miRNAs detected in saliva stains, let-7f-5p, let-7i-5p, let-7a-5p, miR-486–5p and miR- 21–5p were the most abundant expressed miRNAs. Our work may shed light on MPS-based approaches with miRNA analysis of saliva in forensics and provide new way in exploring the miR- NAs in aged or decayed trace samples.

698 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS STANDARDS, QUALITY CONTROL, ACCREDITATION, AND ETHICS

P616 - BRAZIL´S DNA PROFILE DATABASE – ESTABLISHMENT, LEGISLATION AND ACCREDITATION Edna Iwamura1, Eloisa Auler Bittecourt2

1 Federal University of São Paulo UNIFESP, Pathology, São Paulo, Brazil 2 Academia de Policia de São Paulo ACADEPOL, Unidade de Criminalística, São Paulo, Brazil

This study aims to present a brief history of the Integrated Genetics Profiling Database Network (IGPDN) in Brazil, as well as the Legislation that regulates the insertion of genetic profiles of individuals in criminal cases. Also, it offers a non-technical summary – with reference to international comparative information – to discuss developments in historical and political context. Recommendations for laboratories, in particular those conducting DNA tests, as the one from Brazilian Society of Legal Medicine (1999), Laws and Reports of IGPDN were analyzed. Forensic genetics in Brazil, as a tool for solving crimes, started in 1995, when the DNA Laboratory of Civil Police of the Federal District was opened. From 2004, the National Secretariat of Public Security, under the Ministry of Justice, promoted the qualification of criminal expertise, the implementation and adaptation of forensic genetic laboratories in Brazil, as a strategy for structur- ing and modernizing the expertise. Since the IGPDN was created in 2009, efforts to ensure efficient and targeted collection of DNA profiles has been made. Law 12,654, dated 2012, provides the possibility of collecting a genetic profile as a form of criminal identification, and determines that those convicted of a crime committed intentionally, with violence against a person, or for any of the crimes foreseen in Law 8,072, dated 1990, will be submitted, to the identification of the genetic profile. In Brazil, we have about 20,000 DNA profiles in the database, New Zealand 158,000, the United Kingdom 6 million and the United States 13 million. Political context and the law explain why, compared to other countries, Brazil is far below the number of DNA profiles inserted in the databases, despite the historical data of homicides.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 699 P617 - COMPARISON OF THREE DIFFERENT PRETREATMENT METHODS AND STORAGE TEMPERATURE FOR BLOOD RNA EXTRACTION Jiayi Zhang1, Qiannan Xu2, Chengtao Li3, Xiling Liu3

1 Inner Mongolia Medical University, Department of Forensic Medicine, Hohhot, China 2 Wenzhou Medical University, Department of Forensic Medicine, Wenzhou, China 3 Academy of Forensic Science, Forensic Genetics, Shanghai, China

In forensic genetics, the RNA expression profiles of specimens obtained from forensic caseworks can be utilized to identify the biological origins, such as the body fluid identification, and age ranges, etc. Meanwhile, RNA is grossly susceptible to external influences, which can be problematic to obtain ideal RNA expression results because of the low integrity and concentration. Thus, the sample pretreatment and storage can play a pivotal role in forensic applications related to RNA researches. In this study, three different pretreatment methods were evaluated for their effects on RNA quality extracted from peripheral blood, including the RNA yield, concentration and integrity. Results illustrated that the RNA pretreated by the preservation solution and RBC lysis buffer and stored at -80 °C had the best quality. This study can be conductive to perform the downstream experiments, such as sequencing. The method of pretreatment can adopt an appropriate method depending on the sample.

700 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P618 - CONTAMINATION ON THE OUTSIDE OF ALLELIC LADDER CONTAINERS Alina Senst1, Iris Schulz1, Ilona Blank1, Sarah Kron1, Alicia Lange1, Renata Janscak1

1 Forensic Medicine Basel, Forensic Genetics, Basel, Switzerland

Forensic PCR kits consist of components required for amplification and capillary electrophoresis. One component is the allelic ladder with highly concentrated amounts of DNA fragments. A contamination by an even tiny amount of allelic ladder must be avoided, especially when pre-PCR areas could be affected. However, we observed such sporadic allelic ladder patterns in negative controls so that we were eager to find the root cause for this contamination. In addition to a thorough investigation of our own laboratory processes we also investigated the outside of the allelic ladder containers from various PCR kits from three manufacturers, even though we did not expect anything from this check. Startlingly, we found allelic ladder contaminations on all outer sides of the reaction tubes, the lids and in some cases even on the outside of the sealed plastic packaging of the allelic ladder. This was true for kits in use but also for containers that had not been opened yet. When purchasing a new product, we as customers do not expect production-related contamination of the outer surfaces so there is a risk of minimal residues of the allelic ladder being carried over to pre-PCR laboratory areas. We contacted the manufacturers concerned asking for a potential explanation and for a check of their own internal production processes. One of them sent us swabs which they took themselves from their outside of new PCR kit components ready to be shipped to customers. We will present these results as well as our own data from more than 250 samples from 12 different kits and further findings. We will also outline our interims strategy to avoid any contamination by handling new forensic products.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 701 P619 - DEVELOPING LEGAL REGULATION OF FORENSIC DNA‑PHENOTYPING IN HUNGARY Monika Nogel1, Zsolt Pádár1, Czebe András1, Gábor Kovács1

1 Széchenyi István University, Research Center for Forensic Sciences and Criminology, Győr, Hungary

Nearly three decades has passed since DNA typing methods were first used in criminal investigations and trials in Hungary. Traditional DNA forensics uses STRs to match a DNA sample to a suspect or database. However, without database hits these markers cannot help the investigators to solve a crime. A considerable amount of literature has been published on DNA phenotyping in the past few years. These studies argue, that with recent advances in MPS technologies it has become feasible and cost effective to genotype larger marker sets for forensic purposes. Technologies such as EVCs raise questions not only about their technical reliability but also about features of the system in which they are being used. Not surprisingly, both ethical and legal aspects should be taken into account since this technology provides more details about the crime scene samples, about individuals, familiar relationships and biogeographical ancestry, about proprietary data. Law should guarantee the prohibition of genetic discrimination, nevertheless provide information from crime scene to aid in the hunt for suspects. DNA-phenotyping has great potential benefits for criminal justice; however, because of the possibilities for its misuse or abuse, important questions have been raised also about reliability, validity, and confidentiality. Considering of trends and needs for privacy regulation in Europe, research has shown that in Hungary far too little attention has been paid to this problematic issue. This paper intends to show the current legislation and ineluctable future steps in this field.

702 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P620 - DEVELOPMENT OF A COMPUTER TOOL TO BETTER DETECT LOW-LEVEL DNA CONTAMINATIONS France Mailly1, Jean-François Lefebvre1, Josée Houde1, Diane Séguin1

1 LSJML - Laboratoire de sciences judiciaires et de médecine légale, Biology, Montreal- Quebec, Canada

Since the introduction of a new probabilistic genotyping software (STRmix™), the LSJML’s forensic genetics experts are now better equipped to provide statistical probative value for complex DNA results (mixtures, partial profiles) that were previously considered unsuitable for comparison. Contamination events (transfer of DNA from personnel or from a concentrated source to a trace DNA sample) occur despite best practices and are inherent to the sensitivity of the technology. These events can have important consequences: weakening the probative value, false exclusion of a suspect, masking the relevant profile. In some events, the contaminating DNA profile may be very partial. It is therefore critical to develop and use approaches that maximize detection of such events. Our current tool for the detection of contamination is limited in its detection power by two factors: 1) the high number of profiles to be searched in our comparison index,2) the staggered addition of relevant profiles to the index. In addition, very partial profiles cannot be searched because they generate too many candidates to allow effective verifications. The alternative, which consists in a manual comparison of the relevant DNA profiles (contemporaries in the technical chain, including sampling step), is time-consuming, laborious and ineffective. A more appropriate tool that targets relevant profiles and uses all available genetic information will quickly detect a large proportion of contaminations, even in the case of a partial minor contributor in a DNA mixture sample.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 703 P621 - DNA TRANSFER BETWEEN EVIDENCE BAGS DURING CASEWORK Claire Mercer1, Damien Abarno1,2, Phillippa Hearnden2, Adrian Linacre1

1 Flinders University, College of Science and Engineering, Adelaide, Australia 2 Forensic Science SA, Biology, Adelaide, Australia

Inadvertent transfer of DNA from the exterior of an evidence bag to other items provides the opportunity for exhibit contamination. It is assumed that evidence bags are DNA transfer vectors, but research which details the extent of this transfer is limited. With increased DNA profiling sensitivity, there is the increased chance of detecting DNA from a contamination within a casework sample. To provide insight into levels of DNA which accumulate on evidence bags through handling and storage during casework, the exterior of evidence bags were sampled. Casework exhibit packages were sampled before and after the examination of the exhibit inside. Mock evidence bags were sampled after 1, 4 and 8 weeks of storage and throughout a process which replicates realistic exhibit handling. DNA quantities recovered from casework exhibit packages were highly variable and produced highly complex profiles, containing many contributors. Although not statistically significant, DNA quantities and minimum profile contributors were higher from most samples taken after exhibit examination, indicating that during examination DNA is added to the surface of the bag. It was observed that through the process of packaging and unpacking an exhibit, DNA from the item was transferred to the bag exterior. The complexity of DNA profiles produced from mock exhibits increased with time of storage. Research into the levels of DNA accumulating on evidence bags enables best practice protocols to be implemented during the handling, transport and storage of casework exhibits.

704 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P622 - EVALUATION OF THE GEDNAP PROFICIENCY TESTS 2016 – 2018 Carsten Hohoff1, Miriam Meyer1, Bernd Brinkmann1

1 Institut für Forensische Genetik GmbH, GEDNAP, Münster, Germany

The GEDNAP proficiency tests for quality control in the field of forensic DNA analysis are carried out under the auspices of the German Stain Commission, the joint commission of Institutes of Legal Medicine and Forensic Sciences in Germany. Participation in the GEDNAP proficiency tests is open to any laboratory, whether a private institute, university institute or governmental laboratory, from any country worldwide. The GEDNAP program is comprised of various modules (i.e., presumptive testing, autosomal, Y- and/or X-chromosomal STR typing, mtDNA sequencing and biostatistical calculations). Usually, more than 200 forensic laboratories -mainly from Eu- rope- participate in each GEDNAP proficiency test. There are two GEDNAP tests per year. A test includes three refence samples and four forensic stains. A certificate is issued by the organising laboratory, stating that the participating laboratory has successfully passed the proficiency test for the specified loci or in a particular module. Here, we present the evaluation of the results from the last three years. A focus lies on the identification of the causes for errors. Typically, there are transcriptional errors, errors caused by over- interpretation of very weak peaks or of stutter peaks. Recent trends will be discussed.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 705 P623 - FIVE-YEAR EVALUATION OF FORENSIC BODY FLUID IDENTIFICATION AND DNA MIXTURE ANALYSIS FROM THE ACCREDITED GHEP-ISFG PROFICIENCY TEST Coro Fernández Oliva1, Julia Garcia-Hirschfeld2, Lourdes Fernández de Simón2, Alejandro Carmona2, Gracia Luque2

1 National Institute of Toxicology and Forensic Sciences, Quality Assurance Service, Las Rozas, Spain 2 National Institute of Toxicology and Forensic Sciences, Biology Service, Las Rozas, Spain

Performing proficiency tests (PT) is an essential component of a laboratory’s quality assurance program. The criteria for the selection of an appropriate PT should rely on the suitability of its scheme with the laboratory routine casework and on the competence of the provider. This competence can be fully demonstrated if the proficiency test is accredited. With 27 years experience as an external quality assurance control, the proficiency program coordinated by the Madrid Department of the INTCF and organized by the Spanish and Portuguese Speaking Working Group of the ISFG (GHEP-ISFG) offers its Basic level as a suitable accredited proficiency test since 2014. This proficiency test, is divided into a kinship and a forensic module. The Kinship module involves three different stains of one component blood and/or saliva (M1, M2 & M3) For the Foren- sic module, one stain of one or two components of blood, saliva or semen (M4) as well as hair (M5) are provided. A theoretical exercise in each module is also offered. In this work we present the evaluation of results submitted by more than 70 laboratories, regarding the forensic DNA mixture item M4 provided in each edition over the past five years, The genetic parameters evaluated were autosomal and Y-chromosome STRs. Body fluid identification evaluation was also assessed. For both genetic analyses and body fluid identification up to 85 % – 100 % of the results were in concordance with the assigned value. Common errors detected regarding the genetic analyses were due to drop out, to drop in, to stutter peaks called as alleles and to a lesser extent a possible contamination of the laboratory. In general, the use of only one presumptive test caused to reach an incorrect body fluid identification.

706 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P624 - FORENSIC DNA PHENOTYPING AND GENETIC GENEALOGY AS PART OF A FORENSIC IDENTIFICATION TOOLKIT Nathan Scudder1,2, James Robertson1, Sally F Kelty3, Simon J Walsh2, Dennis McNevin4

1 University of Canberra, National Centre for Forensic Studies, Canberra, Australia 2 Australian Federal Police, GPO Box 401, Canberra, Australia 3 University of Canberra, Centre for Applied Psychology, Canberra, Australia 4 University of Technology Sydney, Centre for Forensic Science, Sydney, Australia

Operational use of forensic DNA phenotyping and genetic genealogy are advancing to the point of considering these capabilities as part of a broader suite of forensic tools. A recent example is the partnership between a vendor in the United States and their National Center for Missing and Exploited Children to use phenotyping in the context of other forensic disciplines, including anthropology, isotope profiling and pollen analysis [1]. Viewing forensic genomics as part of a wider toolkit draws on the existing concept of forensic intelligence [2, 3]. By combining this with other forensic and non-forensic data, it places forensic genomics squarely into the intelligence stream requiring DNA-based leads to be contextualised in an intelligence paradigm. This allows these capabilities to draw on established law enforcement processes for dealing with uncertainties associated with intelligence, as well as integrate the capability into the intelligence cycle. This approach requires strategies to incorporate forensic DNA phenotyping and genetic genealogy into intelligence product, a different application to traditional forensic DNA statistical analysis. However, such an approach assures these capabilities a place as part of an overarching case strategy. In this context, it can be delivered as a robust capability to help resolve questions of human identity. 1. Parabon Nanolabs, National Center for Missing & Exploited Children to Use Parabon® Snap- shot™ to Aid Identification of Child Victims. 2017. 2. Ross, A., Elements of a forensic intelligence model. Australian Journal of Forensic Sciences, 2015. 47(1): p. 8–15. 3. Ribaux, O. and B.T. Wright, Expanding forensic science through forensic intelligence. Science & Justice, 2014. 54(6): p. 494–501.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 707 P625 - FORENSIC GENETICS IN SEXUAL ASSAULT: A RETROSPECTIVE STUDY ON THE COLLECTION OF EVIDENCE AT THE EMERGENCY DEPARTMENT Pamela Tozzo1, Nespeca Patrizia1, Ponzano Elena1, Caenazzo Luciana1

1 University of Padova, Department of Molecular Medicine, Padova, Italy

The objective of this retrospective study was to examine the accordance between information derived from the written medical report and the results of DNA forensic analyses regarding 122 cases of alleged sexual assault seen at the Emergency Department of Padua Hospital over a period of 5 years. The examination of discrepant results has proved useful to support a broader application of sexual assault management, particularly during the taking of case history. A story of sexual assault with vaginal penetration and ejaculation coincided with the discovery of male DNA using laboratory methods in more than one in every two cases (55 %). Medical reports and laboratory findings were also consistent in the four cases in which the patient reported no penetration; no male DNA was found (3 % of the whole sample). This led to a total of 71 cases (58 % of the whole sample) in which there was a correlation between the victim’s description of the event and the forensic genetics laboratory findings. This study shows a considerable discrep- ancy between laboratory findings and details recorded in medical reports written at the emergency department in the case of victims of sexual assault and our findings suggest that hospital services which deal with victims of sexual assault should pay more attention to the methods used to obtain and record the material collected during the victim’s medical examination, and to interview patients about the episode. In order to understand the dynamics of the episode in rape cases, and to avoid discrepancies between medical reports and legal reconstructions of sexual crimes, it is crucial to provide victims with support, and to maximize their confidence in the healthcare providers who first attend to them.

708 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P626 - IN-HOUSE VALIDATION OF MPS-BASED METHODS IN A FORENSIC LABORATORY Maja Sidstedt1,2, Klara Junker1, Christina Forsberg1, Lina Boiso1, Peter Rådström2, Ricky Ansell1, Johannes Hedman1,2

1 National Forensic Centre, Swedish Police Authority, Linköping, Sweden 2 Applied Microbiology, Department of Chemistry, Lund University, Lund, Sweden

Massively parallel sequencing (MPS) methods are increasingly applied in forensic casework. Pre- viously, validation guidelines for PCR-based workflows and CE-based STR analysis have been reported. Targeted MPS is based on PCR, but also comes with specific challenges concerning in-house method validation. In this work, we discuss measures that need to be taken when validating MPS-based methods in a forensic laboratory. To that end, we describe our validation of the ForenSeq DNA Signature Prep Kit (Verogen) for analysis of ancestry- and phenotype-informative SNPs. Examples of how to set up an in-house validation study, the outcome of our experiments and general considerations for validation of MPS-based methods are presented. When validating the SNP assay, we focused on the reliability of SNP genotype calls and the compatibility with commonly analysed sample types. Other issues, for example analytical thresholds and accuracy of the data prediction model were considered to be covered by the developmental validation of the kit. Our study included: (1) confirmation of correct genotype calls, through analysis of both reference material with known genotypes (2800M and 9947A) and by repeated analysis of other samples, (2) determining limit of detection, applying two independent dilution series and (3) investigating PCR inhibition, by analysing relevant sample matrices. In conclusion, the MPS-based SNP assay showed overall good performance for single-source samples, with correct genotype calls. We welcome a broad discussion on how to perform in-house validation of MPS-based methods, as this is vital to ensure timely implementation of reliable assays in forensic laboratories.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 709 P627 - METHOD DESIGN TO CREATE AND CHARACTERIZE SIMULATED SEXUAL-ASSAULT SAMPLES AND ESTABLISH ITS USEFULNESS TO VALIDATE FORENSIC ANALYSIS TECHNIQUES Patricio Reyes1, Makarena Escobar1, Viviana Salinas2, David Garate3, Jaime Pantoja1

1 Legal Medical Service, Laboratory Department- Forensic Genetic Unit, Santiago, Chile 2 Legal Medical Service, Laboratory Department- Biochemistry and Criminalistics Unit, Santiago, Chile 3 Legal Medical Service, DNA National Registry, Santiago, Chile

Differential extraction is a modified organic extraction method, which separates epithelial cells (EF) from sperm cells (SF). Validation of analysis techniques in forensic laboratories is necessary to ensure the quality of the results. However, the use of real samples to perform this validation is not the most appropriate, for ethical and legal reasons. The present work aims to design a method to create and characterize simulated sexual-assault samples, and to establish its usefulness to validate forensic analysis techniques. For this, a semen sample from an unknown donor was used to quantify sperm number, as well as a blood sample from a female donor. Swabs and cloths sets were prepared: with semen dilutions ranging from 1 to 1/10,000 in the presence and absence of 50ul of blood. This material was analyzed biochemically by p30 detection and optical microscopy. In addition, DNA extracts obtained from the EF and SF were quantified by RT-PCR and amplified by PowerPlex21 kit. The biochemical results showed p30 and sperm presence in dilutions ranging from 1 to 1/1000. The molecular results in all dilutions indicated that, the average amount of non-masculine DNA present in EF was majority and close to 130ng/ swabs. Meanwhile in SF the DNA present was mostly male and was decreasing in the same order of magnitude of each dilution. In addition, was possible obtained % recovered sperm whose lowest value was 13.8 %. Pure profiles of male contribution were obtained in SF electropherograms of dilutions ranging from 1 to 1/10 in swabs and 1 to 1/100 in cloths. As long as, mixed profiles with full allelic recovery for both contributors in dilutions ranging from 1 to 1/1000 was obtained. These results allowed to demonstrate the success of this method and its usefulness in the validation

710 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P628 - MINIMIZING HAND-TO-GLOVE DNA CONTAMINATION Margreet Van Den Berge1, Samantha Wagner1, Erin Meijers1, Bas Kokshoorn1, Ate Kloosterman1, Mikle van der Scheer1, Titia Sijen1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

In this study, the Netherlands Forensic Institute’s (NFI) contamination prevention recommendations to prevent hand-to-outside-glove DNA transfer were assessed for effectiveness and subsequently optimized. Initial tests were performed using a fluorescent solution that was applied to the hands of volunteers prior to putting on a pair of new laboratory gloves. Visualization using a fluorescent light source showed that contact between the hand and the exterior of the glove is a realistic scenario for various areas of the glove, but predominantly for the wrist area. After examining the extent to which cellular material (DNA) was transferred to the outside of the glove (by sampling gloves without fluorescent solution), attempts were made to reduce this potential contamination risk. For this, the effect of a cleaning protocol with various types of decontamination reagents on a donned glove was assessed. After subsequently confirming that the use of these chloride-based solutions does not have a negative effect on the amount and quality of DNA obtained from traces of evidence upon glove contact, the contamination prevention recommendations were updated. The results of this study support the choice of using a 0.3 % sodium hypochlorite solution or commercially available RNase AWAY as decontamination reagents to clean and subsequently dry the exterior of donned gloves prior to entering the lab and/or handling items of evidence.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 711 P629 - NIST SCIENTIFIC FOUNDATION REVIEW ON DNA MIXTURE INTERPRETATION John Butler1, Hari Iyer2, Rich Press1, Melissa Taylor1, Peter Vallone3, Sheila Willis1

1 National Institute of Standards and Technology, Special Programs Office, Gaithersburg- Maryland, USA 2 National Institute of Standards and Technology, Statistical Engineering Division, Gaithersburg- Maryland, USA 3 National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg- Maryland, USA

The U.S. National Institute of Standards and Technology (NIST) has been Congressionally-funded to perform scientific foundation reviews of select forensic disciplines. These reviews are intended to establish what is well-known and well-supported empirically in a forensic field and identify gaps that need further study. DNA mixture interpretation was selected as the initial NIST scientific foundation review given the existence of abundant literature and a need expressed by members of the community. Multiple interlaboratory studies conducted by NIST and others have noted variability among accredited laboratories using validated approaches on the same DNA mixture data. A six-member NIST review team has conducted this review with input from a Resource Group composed of 13 experienced practitioners and researchers. More than 600 articles related to DNA mixture interpretation have been gathered and examined to understand capabilities and limitations as reflected in the scientific literature. An important goal of this project is to identify, consolidate, and share core principles and supporting publications with the community to encourage deeper learning and understanding of DNA mixture interpretation. The report from this review, entitled DNA Mixture Interpretation: A NIST Scientific Foundation Review, will be initially published in draft form so that community feedback can be considered.

712 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P630 - ON THE ROAD - ACCREDITATION OF FORENSIC DNA LABORATORIES AS A PART OF THE “EFSA 2020” CONCEPT Monika Nogel1, Zsolt Pádár1, Gábor Kovács1

1 Széchenyi István University, Research Center for Forensic Sciences and Criminology, Győr, Hungary

In 2011 the EU Council established the Vision for European Forensic Science 2020 including the creation of European Forensic Science Area by 2020 (EFSA2020). The document highlights, it is essential “to define commonly accepted minimum forensic science standards for the collection, processing, use and delivery of forensic data relating inter alia to data concerning DNA profiles, as well as dactyloscopic and other biometric data, and to equip the Union to meet the new challenges”. In 2016 the Council approved a new Action Plan in the way forward in view of the creation of EFSA2020. ENFSI’s STEFA (Steps Towards a European Forensic Science Area) project is an important step- ping stone in the realisation of the EFSA2020 contributing to key work streams that have been specified in the relevant EU Council Decisions. There are specific activities within the project, e.g. collaborative exercise covering the forensic disciplines of DNA, powering forensic genetic DNA databases for the interpretation of next generation sequencing profiles, etc. The obligatory accreditation of forensic DNA laboratories on the basis of Council Framework De- cision 2009/905/JHA was also important from the point of view of cooperation among Member States. While 2020 is approaching, various stakeholders and Member States currently implement some of EFSA2020 elements. However, there is abundant room for further progress in creating the common forensic area. This paper will focus on results of Hungary and the other V4 countries and the path forward in this field.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 713 P631 - PAST, RECENT AND FUTURE HUNGARIAN LEGISLATION OF FORENSIC DNA ANALYSIS Monika Nogel1, Zsolt Pádár1, András Czebe1, Gábor Kovács1

1 Széchenyi István University, Research Center for Forensic Sciences and Criminology, Győr, Hungary

The Hungarian law sets the conditions approved to perform DNA analysis and the same time es- tablishes national database of DNA report. The legal framework composes Act XLVII of 2009 on the Criminal Records System on the Records of EU Member State Court Rulings against Hun- garian Citizens and on the Records of Biometric Criminal and Law Enforcement Data, Act XXIX of 2016 on Judicial Experts, Act CXII of 2011 on the Right of Informational Self-Determination and on Freedom of Information, Regulation 12/2016. (V.4.) of Minister of Interior on rules of fingerprints, palm prints, taking photographs and DNA samples, Provisions of 31/2008. (XII. 31.) of Minister of Local Government on the work of forensic experts. Regulation of the European Union must be considered, as well. One of the most important boosts undertaken by the EU in recent years on combating transnational crime has been the development of the Prüm Treaty and its partial transformation into an EU-wide cooperation tool under Council Decision 2008/615/JHA and Council Decision 2008/616/JHA. The General Data Protection Regulation (GDPR) has also introduced a new and seemingly quite far-reaching ex- emption to the general prohibition for the processing of genetic data. The question of whether and which kind of consent is required remains is left to other applicable EU law and national laws. The aim of this study is to provide an overview on how the Hungarian legislation of forensic DNA have changed in the past 27 years, as well as to show the current legal context and to provide a conceptual theoretical framework for future legislation, including the rules of forensic DNA typing and the legal regulation connected to DNA-databases.

714 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P632 - STANDARDIZATION IN FORENSIC GENETICS AS A MULTI-FACETED CHALLENGE IN POLAND Renata Jacewicz1, Sasitaran Iyavoo2

1 Medical University of Lodz, Medical and Forensic Genetics, Łódź, Poland 2 Anglia DNA Services, Scottow Enterprise Park, Norwich, United KIngdom

Extremely dynamic development of forensic genetics in the 21st century requires cooperation of the professional bodies and institutions in order to make the best use of technological progress for the public good. The need of high quality of DNA testing for justice and law enforcement in Poland forces the necessity of standardization in this area based on normative and legal requirements existing in Poland and throughout the European Union. Among them the crucial is the requirement of accreditation in accordance with the ISO 17025 standard with the fulfillment of the requirements of DAB-10 document. Regardless of this, an important factor guaranteeing the competence of research personnel is the specialization in the field of laboratory forensic genetics, as well as carry out their work in compliance with good laboratory practice principles (GLP). In the context of standardization a particularly significant are also research guidelines and recommendations that are developed and published by Teams of Experts considering their professional experience as well as widely recognized international recommendations. In conclusion harmonization in forensic genetics in Poland is a multi-faceted challenge that is implemented taking into account the interaction of all mentioned factors, their aims to provide high-quality genetic research in forensics, both in personal identification as well as kinship determination.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 715 P633 - STANDARDIZING DNA-REPORTING ON ACTIVITY LEVEL: A CHALLENGING OPPORTUNITY Nick Laan1, Nathalie Raats1

1 Netherlands Register for Court Experts NRGD, Ministry of Justice and Security, Utrecht, Netherlands

The Netherlands Register of Court Experts (NRGD) guarantees, promotes and monitors the quality of experts in the legal process. DNA-source level is a field of expertise which has been thoroughly identified and demarcated by the NRGD. However, this is not the case for DNA-activity level. Currently, judges, lawyers and prosecutors are hesitant to request activity reports. In contrast to the increasing interest to evaluate DNA evidence using propositions at the Activity Level. The demand is very likely to increase if there is more awareness amongst the courts on what DNA activity reporting exactly is and what its evidentiary value could be. Therefore, the NRGD is currently working on the demarcation of DNA-activity level as a field of expertise by defining the quality standards for the experts. To do so, a DNA Activity Expert Meeting was organized by the NRGD in January 2018. Representatives of various stakeholders were present to discuss the feasibility of standardization of DNA-activity level reporting as a separate competency standard for registration. This expert meeting resulted in a SWOT-analysis of standardizing DNA-reporting on activity level (strengths, weaknesses, opportunities and threats). There was general consensus on the positive impact DNA-activity level reporting could have in the courtroom as it can increase awareness concerning the difference between source and activity level. Further- more, DNA-activity level reporting could help the court understand the limitations of source level conclusions, to help them formulate more relevant questions and help the investigation further. Finally, DNA-activity level reporting could help provide for a better assistance of the courts and lead to better informed decisions.

716 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P634 - STRNaming: STANDARDISED STR SEQUENCE ALLELE NAMING TO SIMPLIFY MPS DATA ANALYSIS AND INTERPRETATION Jerry Hoogenboom1, Kristiaan van der Gaag1, Titia Sijen1

1 Netherlands Forensic Institute, Biological Traces, The Hague, Netherlands

In recent years, various groups have proposed a variety of nomenclature systems for Short Tan- dem Repeat (STR) alleles analysed with Massively Parallel Sequencing (MPS). These nomenclature systems require a centralized organisation to coordinate and standardise the naming at each STR locus. Here, we propose an independently-functioning solution, publically available online and offline, to automatically assign a unique, human-readable, sequence-descriptive name for any STR sequence at any locus in order to provide a universal STR sequence allele naming format for presentation of data. Our proposed algorithm, STRNaming, is only guided by an assigned reference sequence at a locus and automatically decides what the relevant repeat unit(s), the flanking and the intervening sequences are and at which positions the repeat structures start and end. The main goal in the design of this STR naming tool was to derive short and human-interpretable names befitting forensic practise, which means that not only the capillary electrophoresis allele number is derived, but that it is also ensured that a small change in the sequence results in a correspondingly small change in the allele name. Sequence variants outside of the repeat units are indicated as simple variant calls from the assigned reference sequence. The algorithm can even efficiently shorten complex STR loci like those consisting of two separated STR units, by reporting only the deviations from the reference sequence (if any) in the region between the two STR units. Because STRNaming is fully guided by an assignable reference sequence, no central coordina- tion or configuration is required and the method will work for any STR locus be it autosomal, Y-, X-chromosomal in current or future use.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 717 P635 - THE IMPACT OF FORENSIC GENETICS ON THE MANAGEMENT OF SEXUAL ASSAULT VICTIMS: A MULTICENTRE GE.F.I PROJECT Sarah Gino1,2, Marco Bo3, Rossana Ricciardelli4, Milena Alù5, Ilaria Boschi6, Eugenia Carnevali7, Matteo Fabbri8, Paolo Fattorini9, Andrea Piccinini10, Carlo Previderè11, Andrea Verzeletti12, Pamela Tozzo13, Luciana Caenazzo14

1 Laboratory of Criminalistic Sciences “Carlo Torre”-, Department of Public Health and Pediatrics- University of Turin, Turin, Italy 2 Department of Health Sciences, University of Piemonte Orientale, Novara, Italy 3 Legal Medicine Unit, Local Health Trust TO5, Chieri TO, Italy 4 Settore Medico Legale-, Direzione Generale- Azienda Ospedaliero-Universitaria, Parma, Italy 5 Laboratory of Forensic Genetics, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy 6 Laboratorio di Genetica Forense- UOC Medicina Legale-Direzione Strategica, Fondazione Policlinico Universitario Agostino Gemelli, Roma, Italy 7 Laboratorio di Genetica Forense- Struttura Complessa Universitaria Medicina Legale, Azienda Ospedaliera Santa Maria, Terni, Italy 8 Laboratory of Immunology and Forensic Genetics- U.O.L. of Legal Medicine, Department of Medical Sciences, University of Ferrara, Ferrara, Italy 9 UCO of Legal Medicine, Department of Medicine- Surgery and Health- University of Trieste, Trieste, Italy 10 Forensic Genetics Laboratory, Department of Biomedical Sciences for Health- University of Milan, Milan, Italy 11 Laboratorio di Genetica Forense- Unità di Medicina Legale e Scienze Forensi “Antonio Fornari”, Dipartimento di Sanità Pubblica- Medicina Sperimentale e Forense- University of Pavia, Pavia, Italy 12 Legal Medicine Service, Department of Medical and Surgical Specialties- Radiological Sciences and Public Health- University of Brescia, Brescia, Italy 13 Laboratory of Forensic Genetics, Department of Molecular Medicine, University of Padova, Padova, Italy 14 Laboratory of Forensic Genetics, Department of Molecular Medicine-, University of Padova, Padova, Italy

We conducted a retrospective study to analyse the ways in which sexual abuse was handled by the Italian forensic geneticists in order to delineate common strategies in the management of the collected biological samples. In particular, the results of laboratory analyses were compared with the patients’ reports and the outcome of their medical examination, as recorded on standard evidence collection documents. The purpose of this study is identifying which factors could influence the congruence between what was reported and the typing of male genetic profiles. The project was proposed at the Italian Society of Forensic Genetics and 10 laboratories have joined the study filling in a questionnaire. The questionnaire was divided into three sections: one section was about the characteristics of the centre that assisted the woman. A second part was relative to demographic characteristics of women, features of assault, relationship between women and attackers, time elapsed before the taking in care, behaviour of the woman after the assault and before the medical examination, and injuries reported by the victim. The third one was about the characteristics of the collected biological evidence and the type of forensic genetic analysis carried out.

718 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS We analysed 102 cases that occurred between 2006 and 2017. The analysis of the data here presented highlights that the ability to ascertain the presence of male biological material in the collected evidence is not a technical problem for forensic genetics laboratories but it seems to be influenced by other factors, such as how much time elapsed before being taken into care, actions or other relevant events that have occurred between the assault and the medical examination, and the characterisation of the evidence.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 719 P636 - UNLEASHING NOVEL STR LOCI VIA CHARACTERIZATION OF GENOME IN A BOTTLE REFERENCE SAMPLES Katherine Gettings1, Lisa Borsuk1, Justin Zook2, Peter Vallone1

1 National Institute of Standards and Technology, Applied Genetics Group, Gaithersburg, USA 2 National Institute of Standards and Technology, Bioassay Methods Group, Gaithersburg, USA

The higher level of multiplexing possible with current sequencing technologies encourages adoption of additional STR loci to aid in mixture interpretation [1]. However, characterization of these loci and orientation on the human genome is vital for interlaboratory comparability and databasing. Currently, when a laboratory publishes population data from a locus not previously characterized for forensic use, there is no robust way to verify the locus designation, repeat region format, and fidelity of target. To address this, we have evaluated short- and long-read sequence data generated for reference materials included in the Genome in a Bottle (GIAB) Consortium [2] with the goal of reporting STR sequences for loci which may be of interest to the forensic community. Initially, we have analyzed GIAB data using Marshfield sets of primers (published in [3]), including approximately 500 tetranucleotide microsatellite loci, with STRai- tRazor 3.0 [4]. In the future, this approach can be expanded to include other loci of interest. High-confidence STR sequence data will be made publicly available via GenBank record creation within the STRSeq BioProject [5]. As the cell lines represented in GIAB reference materials are available for purchase, this STR dataset represents a robust method for researchers to confirm targeted loci. [1] Novroski NMM, et al. Forensic Sci Int Genet. 2019 Jan; 38:121–129. [2] Zook J, et al. bioRxiv 281006; doi: https://doi.org/10.1101/281006. [3] Pemberton TJ, et al. BMC Genomics. 2009 Dec 16; 10:612. [4] Woerner AE, et al. Forensic Sci Int Genet. 2017 Sep; 30:18–23. [5] Gettings KB, et al. Forensic Sci Int Genet. 2017 Nov; 31:111–117.

720 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS P637 - VALIDATION OF MISEQ FGX™ FORENSIC GENOMICS SYSTEM TO ACHIEVE ISO17025 ACREDITATION Isabel Navarro-Vera1, Sandra Esteban-Navarro1, Sara Ciria-Abad1, Alba Hernandez-Maza1

1 CITOGEN, FORENSIC GENETICS, Zaragoza, Spain

The use of massive parallel sequencing (MPS) is being incorporated quickly into forensic genetics. Although it represents a much more complex and expensive technology than capillary electrophoresis (CE), its advantages are increasingly evident. To allow the use of the data obtainde by MPS for forensic casework, laboratories are commited theirselves to get an accreditation. CITOGEN is the first private forensic laboratory in Spain in achieving the ISO 17025 for MPS applied to forensic genetics. Here we present the data from the validation of the ForenSeqTM DNA Signature Prep (Verogen) kit run into a MiSeq FGx sequencer (Illumina). The validation was done for the Primer Set A (DPMA), which amplifies 58 STRs (Short Tandem Repeat: 27 autosomal STRs, 24 Y-STRs y 7 X-STRs) and 94 iiSNPs (Identity Informative Single Nucleotide Polymorphism). 200 test were done to get enough data to do sensibility, precission and accuracy calculations starting from different types of samples: blood, bucal swaps, semen and extracted DNA, in different supports: FTA cards, swab, clothes and preservatives. All of them had already been tested by capillary electophoresis before in our lab. To asses sensibility, different amplifications starting from different DNA concentrations (0,25–1 ng) and different FTA punch sizes (0,3–2 mm) were done. Precission studies were done with 1ng DNA concentation and 1 mm FTA punches. The precission values obtainded were >98 % for the STRs and >99 % for the SNPs. Accuracy data obtained were over 99 % for both STRs and SNPs.

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 721 722 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Author Index

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 723 Aarts Bart 80 Aloraer Dinah 355 Abad Escala Óscar 265 Alper Behnan 367 Abarno Damien 32, 704 Alsaadi Saleh 90 Abreu-Glowacka Monica 92, 371 Alsafiah Hussain 383 Abu-Qamar Synan 370, 490 Alsalafi Dina 434 Achtruth Sabrina 540 Alsaleh Hussain 306, 642 Acreche Noemí 278 Alshamali Farida 507 Adam Ahmed 308 Alù Milena 65, 145, 718 Adamowicz Michael 611 Aluri Sirisha 79 Aditi Mishra 685 Alvarez-Alvarez Maite 168 Agostini Vincenzo 406 Álvarez-Dios Jose 21, 615, 622 Aguilar-Velazquez Alonso José 271, 283, 318 Alvarez Fondevila Manuel 512, 614, 675, 677 Aguirre Diana 284 Alves Cíntia 76 Ahmed Manzar 110, 209 Amaral Cesar 162, 174, 193–194, 214 Ahn Jun Jae 625 Amaral R. do Marinã 554 Aitthiprajak Pirunpat 505 Ambroa Adrián 21, 512, 615, 622 Aixalà Vidal Ares 265 Ambrosio Brunelli Isabela 279–280, 303, 324–325 Akane Atsushi 141 Ambroziak Jan 212 Akanuma Shigeru 467, 695 Amendt Jens 643 Akhteruzzaman Sharif 650 Ames Carole 504 Akin Cevat 484 Amigo Jorge 675, 678 Akutsu Tomoko 128, 224 Amorim António 76, 225, 237, 314, 328, 349, 386, 584, 601, Alali Abdulkareem 249 607, 609, 681 Al-Ali Fatma 90, 175 An Dan 523 Albani Patricia 53 Andersen Dyrberg Jeppe 19, 633, 639, 654 Albertini Valentina 167 Andersen Meyer Mikkel 58, 397, 594 Albeza Virginia Maria 278 Anderson Florence 73 Albrecht Petra 540 András Czebe 702 Aldosari Waad 217 Andreaggi Kimberly 227, 431 Aler Mercedes 76 Andrea Piccinini 65 Alessandrini Federica 151, 262 Andrekenas Nathália 279, 325 Alexandra Enache 184 Aneli Serena 65 Alfieri Letizia 122 Angelis De Flavio 634 Älgenäs Cajsa 40 An Hyun Sang 429 Alghafri Rashed 190, 277, 308, 311, 370, 375, 468, 477, 685 Anquan Ji 93, 576 Ali Babar 323 An Sangyong 235 Aliferi Anastasia 21–22, 414, 615, 644 Ansell Carina 553 Alin Capilnean 177 Ansell Ricky 465, 709 Ali Qari Absar Muhammad 308 Anselmo Anna 86 Aljanaahi Naeema 490 Anslinger Katja 192, 520 Alketbi Salem 549–550 Antczak Monika 104, 133, 157, 169, 189, 288 Alladio Eugenio 350, 575, 592 Antonio Alonso 380 Almezaina Reem 277 Antunes Marilia 607 Almheiri Maryam 387 Anwar Ijaz 67 Almheiri Reem 190, 308, 311, 477 Aoumtas Sakawrat 505 Almohammed Eida 90–91, 175, 375, 503, 670–671, 693 Aranovich Siarhei 256

724 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Arciszewska Joanna 163–164, 202, 212, 251, 255, 392 Bartáková Karolína 631 Armendáriz-Castillo Isaac 273, 282, 319, 340, 356, 358 Basabe-Desmonts Lourdes 517 Armogida Luigi 249 Basset Patrick 172, 600 Arora Natasha 295 Bastisch Ingo 44, 137, 459, 540 Arroyo-Garcia Rosa 181 Bautista-Gonzalez Enrique Jorge 317, 391 Arroyo-Jiménez Susana 517 Bayer Birgit 192, 520 Arroyo-Pardo Eduardo 96, 138, 185, 265, 301, 326, 415, 482 Beasley Jordan 109 Asaghiar Fisal 475 Beaumont Dan 559 Asawutmangkul Watee 443 Beaumont Daniel 562 Atallah Chahin Valerie 461 Beckmann Britt-Maria 263 Atwood Lauren 17 Beek van der Kees 433 Aune Marthe 287, 432 Bell Michael 17 Aversa Roberta 525 Bemmann Charline 613 Ávila Camilo 269, 276 Bengochea Tatiana 449 Aw Qin Zhen 228 Benito-Lopez Fernando 517 Ay Mustafa 343, 367 Benschop Corina 26, 430, 551, 603 Azuaje-Hualde Enrique 517 Bento Margarida Ana 106, 147, 389, 488 Backx Anouk 26, 551 Bento São Marta 106, 389, 488 Bader Drew 30, 688 Berakovich Gabriela 267 Baerlocher Loic 684 Berger Burkhard 73, 229, 242, 286, 380 Bae Su-Jin 513 Berger Cordula 73, 229, 242 Baeta Miriam 258, 297, 320, 409, 489 Berge Van Den Margreet 80, 673, 711 Baeza-Richer Carlos 96, 138, 185, 265, 326, 415, 482 Berg Gregory 486 Bailo Paolo 167, 406 Berg Thomas 287, 432 Bai Peng 55, 196, 552, 555 Berti Andrea 86, 350, 525, 575, 592 Bajunović Zlatan 532 Bertoglio Barbara 42, 123, 502 Bakri Tracy Mayda 190 Bertolini Emilie 123 Balažic Jože 119, 125, 132, 179, 200, 234, 516, 521 Bertrand Ludes 68 Balding David 58 Bever Robert 110, 209 Ballantyne John 49, 139, 195 Bianca Januário 325 Ballard David 21–22, 61, 73, 113, 121, 156, 186, 414, 598, 644 Bian Yingnan 332–333 Balsa Filipa 76, 106, 389, 488 Biedermann Alex 45 Bamberg Malte 207 Bilić Ana 208, 532 Bandhaya Achirapa 558, 570 Bille Todd 534 Banemann Regine 137 Binali Al Ibrahim 217 Bañón Eladio 320 Bin Cong 230 Barash Mark 650 Bini Carla 65, 151, 448 Barbara Giussy 167 Bintz Brittania 541 Barbarić Lucija 260, 352 Biondi Gianfranco 634 Barbarii Elena Ligia 410 Bird Thomas 254 Barbaro Anna 368, 418, 428, 455, 506 Bitencourt Amanda 162 Barkovits Katalin 676 Bittecourt Auler Eloisa 699 Barni Filippo 350, 525, 575, 592 Bittner Felix 208 Barrio M. Candela 532 Blanco-González Antonio 185 Barrio Pedro 380 Blank Ilona 120, 701 Barritt-Ross Suzanne 63 Blázquez-Martín Paula 305

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 725 Bleka Øyvind 25, 107 Brinkmann Bernd 705 Blumová Barbora 618, 631 Brito Pedro 106, 147, 389, 488 Blythe Martin 441 Bronikowska Agnieszka 16, 20, 653 Boada Anahí 335 Browne Tebah 536 Bochenek Michał 644 Brown K Melanie 88 Bodmer Walter 351, 373 Brustad Kjelgaard Hilde 60 Bodner Martin 61–62, 286, 380, 394, 563 Brynychova Iva 683 Boersting Claus 170 Buadu Sandra 287 Bogale Likie Agajie 404 Buchard Anders 170 Bogas Vanessa 106, 488 Buckel Iris 137 Bogus Magdalena 114 Buckleton John 32, 610 Boiso Lina 709 Budowle Bruce 344, 380, 563 Boiso Samuel 544 Builes José Juan 284, 322, 335, 402 Bo Marco 718 Bunakkharasawat Wanasphon 134, 425 Bonadio Raphael 140 Bunpa Supansa 173 Bonato Omar 102 Buresova Monika 572 Boonderm Nongnuch 396 Burgoyne Leigh 94 Borg Joseph 394 Burrill Julie 51 Borja Trevor 56 Burrows Adria 218 Boroń Michał 16, 20 Buscaino Jacklyn 484 Borovko Sergei 256, 641 Buscemi Loredana 151, 537 Borrego Burillo Luis 76 Buś Magdalena 212, 344 Børsting Claus 19, 337, 374, 388, 526, 639, 654 Busscher Loes 551 Borsuk Lisa 61, 226, 257, 720 Butler John 712 Boschi Beatrice 438, 545, 547 Caba de la Idoia 78 Boschi Ilaria 718 Cabascango Eliana 282 Bosetti Alessandro 123 Cachée Philipp 221 Bottino Carolina 416 Caenazzo Luciana 718 Bourdeau-Heller Jeanne 458 Cainé Laura 681 Bowman Zoe 461 Caixia Li 576 Boyko Toni 80, 219–220 Calabrese Enrica 122 Boyle Rachel 275 Caldeira João Maria 147 Bozzo Ruben Walter 405 Cal de los Ángeles Casares de María 21, 615, 622, 662 Bozzo R. Walter 369 Caliebe Amke 606 Brachold Jaime 152 Calloway Cassandra 427, 457, 538 Braganholi Danilo 279, 325 Campos de los Gustavo 628 Braganholi Faustino Danilo 280, 303, 324 Canan Husniye 182 Brandhagen Michael 115 Candia Di Domenico 167 Branicki Wojciech 16, 20–21, 23, 121, 615–616, 620, 624, 630, Caneparo Denise 592 644, 648, 653 Cannet Catherine 211 Bredemeyer Steffi 374 Cannone Francesco 350 Brenciani Andrea 262 Cano Hortensia 347 Brenner Charles 688 Canteros Malena 369 Brescia Gloria 421, 565, 667 Cao Shuqiang 398 Brestovansky Petr 201 Cao Yueyan 126, 469, 533 Bright Jo-Anne 32, 610 Caputo Mariela 43, 76, 347, 359, 449, 535, 539, 543

726 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Caramelli David 86 Chen Fang 204, 519, 523, 561 Carbonaro Andrea 436 Chen Feng 87, 420 Cardoso Sergio 258 Chengchen Shao 330 Carmen G Morales 463 Cheng Feng 18, 268, 296, 454 Carmona Alejandro 706 Chengjie Ji 576 Carnevali Eugenia 65, 151, 448, 691, 718 Cheng Xiaojuan 298 Carracedo Ángel 512, 615, 624, 678 Chen Jing 422 Carrasco David 181 Chen Lu 230 Carratto Mayra Telles Thássia 635 Chen Man 18, 268 Carvalho Fagundes de Elizeu 274, 321, 390 Chen Peng 87, 420 Carvalho Fagundes Elizeu 162, 174, 193–194, 214, 353, 357 Chen Tong 18 Casadevall Domenech Gemma 265, 326 Chen Xiaogang 469 Casals Ferran 326 Chernomoretz Ariel 310 Casas-Vargas Andrea 289, 502 Cheung Y.Y. Elaine 304 Castagnola Josefina Maria 347 Chew Hui Mee 228 Castella Vincent 172, 600, 684 Chierto Elena 151, 404 Castelli da Cruz Erick 635 Chirnside Olivia 53 Castillo Adriana 338, 353 Chiti Enrica 406 Castoldi Elisa 627 Chi Zhang 93 Castro Aurelio José 278, 290 Choi Dong-ho 429 Catanesi Cecilia 400 Choi Ho Dong 245 Catcheside David 94 Choi Kyoung Sung 245 Catchpole Brian 73, 113 Cho Kuk Hyun 245 Catelli Laura Maria 43, 101, 111, 357 Chong Yin Wai 407, 411 Cattaneo Cristina 42, 123 Choo Minkyu 105, 235 Cavalleri Gianpiero 373 Cho Sohee 171 Cebrián-Fernández Rosario 301 Cho Sop Tae 241 Cecilia Figueroa 463 Chotigeat Wilaiwan 443 Cekin Necmi 343 Choudhary Kumar Sumit 315 Cemper-Kiesslich Jan 178, 239 Christine Keyser 68 Centrone Claudia 302 Chrostowska Magdalena 104, 133, 157, 169, 189, 288 Cepeda Marcela 369 Cicarelli Maria Barretto Regina 280, 303, 324 Cerri Nicoletta 421, 565, 667 Cicarelli Regina 279, 325 Cevik Ekim Filiz 658 Ciccotelli Roberto 627 Chacon-Duran Jazmín Eliana 518 Cihlar Churchill Jennifer 563 Champion Jessica 50 Čílová Daniela 142, 293 Champod Christophe 45, 48, 577 Ciria-Abad Sara 595, 721 Chang Chien-Wei 557 Cisana Selena 592 Chang Joseph 30, 261, 674 Claerhout Sofie 64, 399 Chan Xavier 223, 407 Claes Peter 15 Chen Angela 139 Clarke Jennifer 611 Chen Carol 243 Clayton Tim 37, 88, 559 Chen Chong 95, 160, 210 Cleary John 37 Chen Chuguang 160, 264, 268, 296, 299–300, 398, 454 Colao Emma 65 Chen Dan 196, 689 Coleman Kiera 88 Chen Deqing 408 Colloca Domenico 627

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 727 Colucci Margherita 441 Daniel Runa 17, 619, 650 Concheiro-Guisan Marta 536 Danielson Phillip 413, 529 Conde-Sousa Eduardo 225, 609 Danuphol Suriyanratakorn 396 Cong Bin 117, 259, 365, 548 Dash Ranjan Hirak 682 Conlan Xavier 108, 248 D’Atanasio Eugenia 350 Constantinescu Maria Andrea 410 Dauber Eva-Maria 267 Constantinescu Monica Carmen 410 Dávalos Emilia María 358 Conte Jillian 444, 466 Davenport Lucinda 156, 598 Copeland Sarah 493, 611 Debortoli Guilherme 635 Coppone Mauro 102 Deccache Lara 321, 353 Corach Daniel 43, 76, 347, 359, 449, 535, 539, 543 Decorte Ronny 64, 399 Cordero Maria Ana 265 Defraye Charlotte 399 Cormaci Patrizia 418, 428, 455, 506 Deitos Raphael Alexandre 532 Cornelius Stefan 456 Delabarde Tania 211 Corradi Corradi 385 Dellamico Barbara 75 Corradi Dante 85 Deng Jie 281, 557 Corradini Beatrice 145 Deng Peipei 259 Correa Simoes Dutra Heitor 421, 565, 667 Dente Ângela 237 Correia Ana Maria 532 Derenko V Miroslava 23 Cortellini Venusia 421, 565, 667 Derevianko Anatoly 222 Costa Afonso Heloísa 76, 237, 314, 349, 386, 607 Desmyter Stijn 686 Costas Lòpez Olalla 614 Deus Inés María 310 Costea Catalin Andrei 410 Devesse Laurence 156, 414, 598 Cotts Leonardo 174 Diana Iliescu Bulgaru 177 Courts Cornelius 99, 221 Díaz López Lourdes 76 Court Syndercombe Denise 21–22, 36, 73, 113, 121, 156, 186, Didier Meghan 374, 472, 515 414, 598, 615, 644 Diegoli Toni 534 Couto Cátia 76 Diepenbroek Marta 192, 202, 616 Črešnar Matija 125, 154 Dierig Lisa 527 Crubézy Eric 68 Ding Mei 97 Cruciani Fulvio 350, 575 Dixit Shivani 341, 682, 685 Cuenca Daniela 457 D.Medeiros C. Loyane 554 Cunha Eugénia 237, 314, 349, 386 Doensen Dyon 150 Cunha Patrícia 106, 389, 488 Donadi Antônio Eduardo 635 Curran James 578 Donato Anna 214 Cuscó Ivon 265, 326 Doniec Andrzej 528, 564 Cyplik Paweł 104, 157, 169, 189 Dormontt Eleanor 70 Cytacka Sandra 163, 202, 212, 251, 255, 392 Dorn Annette 676 Czarny Jakub 104, 133, 157, 169, 189, 288 Dorum Guro 49, 195, 605 Czarny Zuzanna 288 Downey Lotte 458, 481, 571 Czebe András 638, 714 Drábek Jiří 181, 618, 631 Dagostino Lucy 650 Drath Joanna 251 Dalihodova Simona 161, 252, 291 Drobac Jon 458, 481 D’Amato Eugenia Maria 74, 218, 316, 354, 694 Dudás Eszter 270 Damji Ruksar 370 Dufva Charlotte 465 Daniel Barbara 51 Duijs Elisabeth Francisca 412, 603

728 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Duijts Liesbeth 663 Fang Chen 698 Dukes Jones Mary 610 Fang Ting 126, 469, 533 Dumache Raluca 184 Fan Qingwei 18, 454 Dunkelmann Bettina 178, 239 Fantinato Chiara 30 Du Qingqing 259, 365, 548 Farag Fathi 478 Dür Arne 348 Farhatullah Muhammad 308 Durdle Annalisa 108, 180 Faris Tom 249 Dwars Elske 673 Fattorini Paolo 42, 65, 123, 151, 448, 516, 687, 718 Eagles Justin 515 Fausser Jean-Luc 68 Eardley Robert 546 Feingold Eleanor 15 Echeverría Albuja Byron 282 Feixat Barrot Carme 116, 144 Eckert Martin 345 Fejes Vivien 206 Edson Suni 486 Felice Alex 394 Eduardoff Mayra 304, 502, 650 Felix F. Janine 663 Egeland Thore 60, 596, 605 Feng Tao 196 Egger Gerda 629 Fernández Juan 96 Ehler Edvard 252, 291 Fernandez-Rodriguez Amparo 78 Ek Linus 544 Fernández-Serrano José 96 Elena Ponzano 708 Fernández-Vilela Andrea 265 Eller J. Ryan 15 Ferragut Francesca Joana 278, 290 Elliot Jennifer 129 Ferreira Gomes Rimarcs 532 Elliott Keith 456, 492, 613 Ferreira Samuel 532, 554 Elsztein Linda 495 Fiallos Gisella 322, 335, 340, 402 England Ryan 382, 417 Fidelis Bugoye 277 Eom Yoon Ki 241 Figueroa Burgos German 127, 269, 274, 276, 471, 497, 518, 560 Erb Daniel 611 Figueroa Carolina Cárdenas Ana 358 Eriksen Svante Poul 59, 216, 364, 602 Filho Gerardo P. Pierre José 554 Erlich Henry 427, 457, 538 Filippo de Cesare 222 Erlich Yaniv 35 Fimmers Rolf 608 Ermakova Natalya 452 Finaughty Chandra 511 Escala Óscar 326 Fioriti Simona 262 Escobar Makarena 710 Fix Carolin 643 Escobar Soledad María 310 Flávia Silva 325 Esparza-Arroyo Ángel 185 Fleckhaus Jan 652 Espinosa Vinueza Diana 326, 426 Fleming Rachel 53 Esteban-Navarro Sandra 595, 721 Fløjgaard Camilla 72 Estrada Mariano 495 Flor Carlo Iannacone De La Gian 372 Etxeberria Francisco 258 Flores Allie 110, 209 Euteneuer Jan 99, 221 Flores-Baas Ismael Rolando 283, 317 Eva Müller 178 Flores-Espinoza Rodrigo 274, 471 Evanoff David 541 Foley Megan 581 Evett Ian 610 Fondevila Manuel 96 Ewing Margaret 571 Fonneløp Elida Ane 47 Fabbri Matteo 65, 102, 122, 151, 718 Forbes Shari 84, 240 Fabiani Martin 374 Forsberg Christina 465, 553, 709 Faccinetto Christian 176, 628 Förster Gabriele 114

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 729 Forsythe Bethany 275 Garcia Gabriela Maria 400 Fracasso Carolina Aguiar Nadia 635 Garcia-Hirschfeld Julia 706 Frances-Cuesta Carlos 78 García-King Antonio Marco 271 Francisco de Oliveira Daniela 470 Garcia Laura Oliveira Ana 633 Franco H. Oscar 663 García Óscar 76 Franken Benjamin 613 García-Rey Sandra 489 Franz Neuhuber 239 Garofano Paolo 575, 592 Frascione Nunzianda 51 Garrett-Rickman Samara 84, 240 Frédéric Grosjean 599 Garrigos Lucía 43, 539, 543 Freire-Aradas Ana 21–22, 512, 615, 622, 624, 644, 650, 662 Garzón-Salazar Alejandra 269, 274 Freire-Paspuel Byron 127, 274, 518, 560 Gastineau Romain 392 Freitas Marcelo Jorge 279 Gaudio Maria Rosa 102, 122 Freyle-Barón Giselle Carmen 497 Gausterer Christian 629 Fridman Cintia 470, 633 Gaviria Anibal 322, 335, 402 Frigieri Isadora 325 Gaviria Aníbal 340 Friš Lina Eva 119, 234 Gehrig Christian 172, 599–600 Frisoni Paolo 102, 122 Geisen Christof 263 Frøslev Guldberg Tobias 72, 74 Ge Jianye 424, 436 Fuerst Angelika 54 Gennari Giuseppe 167 Fu Guangping 117, 230, 548 Genoy-Puerto Alexander 127 Fujii Koji 143, 604 Gentile Fabiano 176, 556, 627–628 Fujimoto Shuntaro 205, 583 Gentile Guendalina 406 Fu Lihong 259, 365, 548 Geppert Maria 394 Funayama Masato 636 Geraut Annie 211 Furfuro Sandra 76 Geršak Miriam Živa 125 Furman Nicolás 369, 405 Gerundino Francesca 302, 438, 547 Fürst Angelika 207 Geryk Jan 683 Gaag van der Kristiaan 26, 551, 590, 686, 717 Getaneh Yimam 404 Gabriel Matt 261, 281, 557 Gettings Katherine 61, 226, 720 Gagliardi Florencia 405 Ghafri Al Rashed 490 Galant Natalia 133 Ghanbari Mohsen 663 Galusha Michelle 498 Giangasparo Federica 73, 113, 414 Gamboa Javier 409 Gibbon E. Victoria 511 Gamboa-Magaña Yaqueline Rosalba 283, 317 Gil Adriana 338 Ganbold Uyanga 393 Gill Peter 25, 47, 107 Gangitano David 116, 144 Ginart Santiago 43, 535, 539 Ganschow Sebastian 453 Gino Sarah 406, 509, 718 Gao Yuzhen 668 Giotti Irene 545 Gao Zehua 126 Girbea Georgiana 410 Garai-Ibabe Gaizka 517 Giriodi Maddalena 406 Garate David 710 Girón-Santamaría Lorena 21, 512, 615, 622, 662 García-Aceves Elizabeth Mayra 318 Gisbert Cuéllar Juli 265, 326 García-Arumí Elena 326 Githae Dedan 121 Garcia Beatriz Sara 639 Giusto Nogueira Di Marina 532 García-Cárdenas M. Jennyfer 273, 282, 319, 340, 356, 358 Gleeson Maurice 37 García Celia 320 GmbH Qiagen 621

730 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Goecker Zachary 56 Guerrero Santiago 273, 282, 319, 340, 356, 358 Gomes Catarina 328 Guevara-Ramírez Patricia 273, 282, 319, 340, 356, 358 Gomes Cláudia 96, 138, 185, 265, 301, 326, 415, 482 Guimarães de Fátima T. Maria 532 Gomes Iva 328, 681 Guo Dan 264 Gómez Beatriz 415, 482 Guo Jiangling 298 Gómez-Tato Antonio 21, 615, 622, 662 Gusmão Leonor 274, 289, 321, 328, 338, 353, 357, 390, 601, 609 Gonçalves de Toledo Fernanda 470, 633 Gutiérrez-González de Pilar Carolina 318 Gondenberg-Barbosa Rodrigo 194 Gysi Mario 295 Gonzalez Angéla 68, 211 Haaf Thomas 630 González Astudillo Odalis 273, 356 Haas Alexandra 510 Gonzalez-Candelas Fernando 78 Haas Cordula 49, 71, 79, 195, 199, 295 Gonzalez-Herrera Lizbeth 283, 317, 391 Haase Tabea Hannah 526 González Montiel Diego 27, 71, 433, 666 Haddish Kiros 404 Gonzalez Nicte Lopez Paola 391 Haddrill Penelope 642 Goodwin William 190, 217, 355, 376, 383, 434, 476, 549–550 Haddrill R. Penelope 213, 306 Gopalakrishnan Anupama 458, 481 Hadi Sibte 375, 476, 693 Gopinath Siddhita 436 Hadrys Thorsten 54, 207, 540 Gordon Rachel 457 Ha Ho Eun 625 Górka Aleksandra 104, 133, 157, 169, 189, 288 Haidar Mahdi 213, 306, 387 Gosch Annica 99, 221 Haizel Thomas 477, 501, 697 Goto-Yamamoto Nami 181 Hallast Pille 188 Gouveia Nair 106, 147, 389, 488 Hall Diana 640, 684 Grabe J. Hans 663 Hamano Yuya 583 Grabmüller Melanie 83, 231 Han Gyun Chang 241 Graham Erica 458, 481 Han Haijun 264 Granda David Juan 497 Han Hee Seong 253 Granda Villacrés Irina 471 Hannig Jan 34 Granizo Eva 258, 297, 409 Hansen Johannes Anders 72, 74 Granja Rafaela 36, 41 Hanson Erin 49, 139, 195 Gratão Marina 532 Han Songho 235 Gray Emma 108 Hao Ting 298 Grdina Sara 119, 234 Hara Masaaki 143, 336 Green Henrik 40 Harbison Sallyann 53, 275, 382, 417, 580, 645 Green Robert 424, 546 Harihar Prakash Sathya 674 Grießner Ines 178, 239 Haring Gregor 179 Grignani Pierangela 42, 65, 123, 151 Harteveld Anne 417 Grini Annalisa 172 Harthun Maria 582 Grosjean Frederic 172, 600 Hartless Sophie 447 Groß E. Theresa 263 Hartman Dadna 461, 650, 664 Gross E. Theresa 531 Hartmann Katharina 643 Gross Michaela 540 Hasap Laila 443, 542 Gross Theresa 540, 646, 650 Hasegawa Ryo 30, 281, 557, 674 Gruezo Carmen 322, 335, 340, 402 Hashiyada Masaki 141, 636, 650 Grzybowski Tomasz 16, 20, 23 Hearnden Phillippa 704 Guan Xueting 103, 636 Heathfield Laura 238, 511 Gudievskaya Irena 641 Hecht Werner 229, 242

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 731 Hedman Johannes 553, 617, 655, 709 Hornik Bartosz 104, 133, 157, 169, 189, 288 He Guanglin 312–313, 378 Horres Ralf 643 Heidegger Antonia 14, 612, 620, 630, 650 Hortelano-Uceda Ignacio 301 Heinrich Josephin 73, 229, 242 Hosseini Mostafa Sayed 656 Heitmann Ingebjørg 47 Houde Josée 703 Hellmann Andreas 229, 242 Houshdaran Sahar 629 Helm Katharina 178, 239 Houston Rachel 116, 144, 462 Hempel Jamie 166 Hou Yiping 155, 197, 312–313, 334, 374, 378, 597, 690, 696 Hendricks Kayla 493 Hsieh Hsing-Mei 232 Henry Julianne 423, 566, 578, 650 Huang Lei 333 Henry Solar 463 Huang Sicheng 573 Hens Greet 15 Huang Yuguo 126, 469, 533 Herman Renata 133 Huang Yun 165, 451, 485 Hermanson Spencer 571 Huber Gabriela 191, 563 Hernández-Cordero Ariana 415, 482 Huber Nicole 348, 394, 563 Hernandez-Maza Alba 595, 721 Huel René 150, 208, 532 Hernández-Sierra del Pilar María 236 Hughes-Stamm Sheree 462 Hernandis Elias 605 Hu Hui 360 Herrasti Lourdes 258 Huidobro Victoria Lareu María 512, 614, 662, 675, 677 Herrera-Diaz Guadalupe Refugio 283, 317 Hulaniuk Laura Maria 347 Herrera J Rene 297 Hu Sheng 124 Heß Aurora Sarah 149 Huszar Tunde 351 He Wang 689 Hutnik Katarzyna 351, 373 He Yan 668 Hu Yuhan 126, 469, 533 Hickey Stephanie 472 Hwang Kwan In 626 Hicks Tacha 45, 48, 172 Iacob Diana 54 Hill Kelly 423, 566 Iadicicco Agata 42, 123 Hill Tim 515 Ibarra Adriana 276, 497 Hirai Eriko 205, 583 Idkowiak Joanna 104, 133, 157, 169, 189, 288 Hochstein Norbert 613 Idrizbegović Šejla 150, 208 Hohoff Carsten 14, 705 Igari Yui 103, 636 Holicki Mariusz 251 Ikram Arfan M. 663 Holinková Veronika 618, 631 Ilina Viktoriya 586 Holländer Olivia 659 Indencleef Karlijne 15 Holland M. Mitchell 499 Inglez Mariana 532 Holmer Anders 526 Ingold Sabrina 49 Holt Allison 129, 139, 484 Inkret Jezerka 179, 200 Holt Cydne 374, 472, 515 Inokuchi Shota 604 Honda Katsuya 467, 695 Inturri Serena 406 Hongmei Xu 330 Ioganson Elena 135 Hong Rom Sae 626, 650 Iozzi Sara 438 Hongxia He 89 Irwin Jodi 29, 115 Hoogenboom Jerry 430, 551, 590, 603, 717 Isao Yokota 128 Hoogendorp Nathan 590 Ishak Insyirah Binte Nurul 228 Hopkins Catherine 423, 566, 578 Ishak Sarah 70 Hopwood Andy 481 Ishii Akira 679

732 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Ishizawa Fujio 467, 695 Jin Jong Kim 429 Iwabuchi Yayoi 467, 695 Jobling A. Mark 36, 188, 213, 351, 373, 441 Iwamura Edna 699 Johannessen Helen 47 Iyavoo Sasitaran 476–477, 501, 697, 715 Johann Schulze Kristina 244, 608 Iyengar Arati 376 Johnston Emma 158 Iyer Hari 33–34, 712 Jones Richard 373 Iyer Hariharan 257 Joo Hyunjung 105, 235 Jacewicz Renata 715 Josefina T Honorato 463 Jacie Wu 493 Joudaki Atefeh 294 Jacobs Werner 479, 483 Jung Mok Sang 253 Jacobs Zenobia 222 Jung Saimi 429 Jaddoe W.V. Vincent 663 Jung Sang-Eun 626 Jager Dawn De Megan 74 Junior Carneiro da Silva Ronaldo 342, 346 Jäger Richard 149 Junior Teixeira Mendes Celso 635 Jagielska Zofia 494 Junker Klara 617, 655, 709 Jaime Astudillo Pantoja 463 Just Rebecca 29, 498 Jakovski Zlatko 460 Just S. Rebecca 107 Jałowińska Katarzyna 163, 202, 255, 392 Kaczorowski Łukasz 104, 133, 157, 169, 189, 288 Janeczko Jarosław 630, 644 Kaitholia Kamlesh 682 Janevski Robert 384, 460 Kakimoto Yu 649 Jankova Renata 384, 460 Kalamara Vivian 657 Janosch Andrea 613 Kalaoglu Esen 367 Janscak Renata 701 Kalkovaliev Branko 384 Janssen Kirstin 287, 432 Kampmann Marie-Louise 374 Jansson Linda 553 Kandratsiuk Alexander 641 Januario Belon Bianca 279–280, 324 Kang Kelai 93 Januário Belon Bianca 303 Kang Minhye 235 Januła Miłosz 528, 564 Kang Pil-Won 241 Jarosz Agata 644, 653 Kang Won Pil 626 Järving Reet 487, 500 Kanokwongnuwut Piyamas 442, 450, 491, 567 Jasinski Edward Marek 164 Kapema Bupe Kapema 285 Jede Aline 582 Karłowska-Pik Joanna 16, 20 Jehaes Els 479, 483 Karlsson Emilia 465 Jeikal Bongjin 105, 235, 253 Kastinger Tamara 178, 239 Jeon Bin Won 253 Kasu Mohaimin 316, 694 Jerman Ivan 154 Katsara Maria-Alexandra 648 Jia Li 160 Kauferstein Silke 263 Jiang Youjing 661, 665 Kawai Yosuke 679 Jian Hui 300, 579, 669, 672, 692 Kayser Manfred 12, 14, 27, 71, 433, 620, 624, 630, 648, 657, Jianhui Xie 330 663, 666 Ji Anquan 89, 124 Keckarevic Dusan 292, 439 Jian Ye 89, 576 Kecmanovic Miljana 292, 439 Jimenez Celia 138 Kelty F Sally 39, 707 Jiménez Planterose Benjamin 666 Kennedy Finlay 559, 562 Jiménez Susana 305, 320 Kennett Debbie 36–37 Jin Bo 661, 665 Kenny David 146

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 733 Ken Watanabe 128 Knights Simon 501 Kerr Zane 32 Knijff de Peter 374, 433, 551 Keshinroe Sam 357 Knijff De Peter 26, 28 Keshinro O Sam 328 Koch Guillaume 211 Kessler C.M. Maitê 554 Kohns Annika 456 Keyser Christine 211 Koichi Sakurada 128 Khan Farhan Muhammad 323 Kokshoorn Bas 80, 219–220, 711 Khan Ramzan Muhammad 308 Kolb Julia 459 Khodeneva Natalya 452 Kollmann Dagmar 267 Khoory Sarah 277 Konarzewska Magdalena 371, 419, 494 Khubrani M. Yahya 213 Kongmeka Nattakarn 173 Khubrani Yahya 188, 383 König Margaretha 456 Kibayashi Kazuhiko 508 Korabecna Marie 683 Kidane Eleni 404 Korolija Marina 260, 352 Kidd Kenneth 30, 650, 674, 688 Kotkin Melissa 472 Kiesler Kevin 226–227 Kotková Lucie 618, 631 Kilchevsky Alexander 641 Kovačević-Grujičić Nataša 23 Kim Hye Eun 241, 429 Kovács Gábor 638, 702, 713–714 Kim Hyeon Gyeong 130 Kozlikin Maxim 222 Kim Il Man 241 Kozma Zsolt 206 Kim In Hye 625 Kraaijenbrink Thirsa 28 Kim Jin-Young 245 Kraaij Robert 71 Kim Ji-Young 118 Krassotkin Yevgeniy 586 Kim Kijeong 159 Kratzer Adelgunde 295 Kim Kyungyong 159, 464 Kravtsova Olga 135 Kim min Bo 379 Kreindl Gabriele 178, 239 Kim Min-Hee 130 Kreutzer Susanne 79 Kim Namyul 105, 235 Kron Sarah 120, 701 Kim Soo Eung 241 Kruijver Maarten 32 Kim Sook Hyo 241 Kruzic Ivana 499 Kim Soo Tae 253 Kua Wei Guo 223 Kim Sungmin 131 Kubinyi Eniko 293 Kim Taegyu 105, 235 Kuhlmann Stephan 676 Kim Ye Nam 626 Kukla-Bartoszek Magdalena 16, 20, 23, 616, 653 Kim Yeon Ga 253 Kulstein Galina 207 Kim Young Moon 171 Kumar Hareesh 404 King Jonathan 61 Kumawat Ramkishan 341, 685 Kirkbride Paul 442, 450, 524, 567 Kundu Biduth 507 Kitpipit Thitika 173, 443, 505, 542 Kunin Victor 135 Kjærbye Bruun Anne 639 Kunze Sonja 663 Kleinbielen Tamara 236, 297 Kupiec Tomasz 403, 480, 528, 564 Kling Daniel 38, 362, 588, 596 Kurganov Karim 587 Klippmark Therese 40 Kurganov Sardarxodja 587 Kloosterman Ate 711 Kurka Grzegorz 251 Klungjan Wasananan 173 Kustermann Alessandra 167 Knap Agata 120 Kwok Ross 146

734 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Kwok Yan Foong Rachel 136 Lee Young Eun 379 Kwon Ye-Lim 379, 513 Lee Young Hwan 625–626, 650 Laan Nick 716 Lee Yu Jun 228 Labaj Pawel 121 Lefebvre Jean-François 703 Lackey Angela 129, 152, 281, 424, 436, 557 Leijnen Gitte 479, 483 Lagacé Robert 30, 49, 198, 281, 433, 557, 563, 650, 674 Leire Ruiz 168 Lago Giampietro 556 Le Jennifer 29, 107, 498 Lai Michelle 223 Lekube Xabier 168 Lall Gurdeep 373 Lemalu Anna 53 Lampert Jodie 373 Lemos Carlos Manuel 349 Lancaster Holly 504 Lenz Kristy 514 Lancia Massimo 691 Leo De Domenico 395, 440 Landry Roxane 397 Leon-Acosta Fabiola Stefany 283, 317 Lange Alicia 701 Leonard Michelle 37 Lang Min 690 Leon De Christina 56 Lao Oscar 71 Leone E. Paola 273, 282, 319, 340, 356, 358 Lareu Victoria María 21–22, 304, 615, 622, 624, 644, 650, 678 Lesaoana Mpasi Motsoboeana 316 Lari Anna 438 Leskovar Tamara 154 Larionova Elmira 452 Le Wang 93, 576 Larmuseau H.D. Maarten 64 Liang Weibo 55, 87, 196, 233, 299–300, 398, 420, 552, 555, 573, LaRue Bobby 116 579, 661, 665, 669, 672, 689, 692 Lee Candy 228 Liberty Amy 261 Lee Deok Soong 171 Li Bo 222 Lee Eun Ji 626 Li Caixia 124 Lee Han-Seong 235 Li Chen 160, 268, 296, 454 Lee Han Yang 241, 626 Li Chengtao 95, 100, 210, 329, 332–333, 360, 474, 700 Lee Hee Eun 625 Li Haixia 331, 381, 437, 593 Lee Hyeon Hye 241 Li Hang 299 Lee Hyun Ji 625 Li Jiang 576 Lee James 266 Li Jinlin 333 Lee Jin Hye 118 Li Li 366, 665 Lee Jung Eun 241 Li Lijuan 668 Lee Keun Myoung 15 Lim Gui Zi 136 Lee Min Jeong 625 Lim Han Hong 570 Lee Mu-Yeong 429 Lim Holden 223, 407 Lee Myeong Jin 245 Li Min 332–333 Lee Seung Han 105, 253 Lim Jessica 281, 427, 557 Lee Sheng Yong 228 Lim Wei Siong Holden 411 Lee Sub Dong 626 Lim Wen Xiang Wilson 136 Leeuw de Rick 374, 551 Linacre Adrian 50, 70, 442, 450, 491, 496, 524, 567, 704 Leeuw de Rick 26, 28 Linarello Pasquale 406 Leeuwen van Rachel 673 Linden van der Jennifer 430 Lee Yeon Chang 253 Lindskou Mads 602 Lee yeon Ji 679 Lin Yuan 95 Lee Yongjiu 183 Lipovac Korana 352 Lee Yoon Jung 429 Li Ran 437

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 735 Liryo Andersen 532 López-Parra María Ana 96, 138, 185, 265, 326, 415, 482 Li Shujin 117, 230, 259, 365, 548 Loreille Odile 115 Liu Baonian 52, 574 Los Marta 419 Liu Fan 663 Loten Mary 458, 481 Liu Hai 197 Loureiro Sara 681 Liu Jiajia 82 Lovisolo Flavia 509 Liu Jinding 298, 408 Lowe Andrew 70 Liu Jing 197, 334 Lucenti Elena 102 Liu Kuo-Lan 232 Lu Chaolong 365, 548 Liu Liu Yuqing 672 Luciana Caenazzo 708 Liu Qingxia 548 Luciel Alma Rincon 284 Liu S Jonathan C 493 Ludeman Matthew 436, 484, 546 Liu Wenli 698 Ludes Bertrand 211 Liu Xiling 100, 474, 700 Luhauniou Arthur 256, 641 Liu Xu 18, 698 Lundqvist Bo 40 Liu Yacheng 160, 268, 296, 454 Lunn Maria Birk Maja 639 Liu Yacheng Yacheng 82 Luo Haibo 165, 451, 485 Liu Ying 82 Luo Lidong 493 Liu Yuqing 300, 398, 552, 555, 579, 669, 692 Luo Xiaoying 661, 665 Liu Zhiyong 18, 268, 296 Luque Gracia 706 Livesey Michelle 354 Luque Juan 585 Li Vivienne 246 Lutz Roewer 13 Li Wanshui 422 Lv Meili 573, 579, 661, 669, 692 Li Yifan 160, 264, 299–300, 398 Lydie Samie 599 Li Yingbi 451 Lydie Samie-Foucart 600 Li Yuan 576 Lynch Courtney 53 Li Zheng 87, 420 Maas C.E. Silvana 663 Li Zhilong 55, 196, 233, 552, 555, 661, 665 Machado Helena 41 Lobato Ester 320 Machado Paulo S. Marcos 532 Lobo Y.A. Rubiane 554 Machalski Grzegorz 251 Locarno Laura 85, 385 Machida Mitsuyo 508 Lombardi Laura 525 Macphetridge Ann 514 Longaray Micaela 111 Macur Aneta 630, 644 Long Bin 689 Madea Burkhard 83, 231 Loockerman Coral 462 Madero Moncada Julie 289 Loo Shuzhen Eileen 647 Magaña-Loarte Concepción 96 Lopes Virgínia 106, 389, 488 Ma Guanju 548 López-Carrera Marcelo 518 Mailly France 703 López-Cortés Andrés 273, 282, 319, 340, 356, 358 Makam Harry 611 López Díez Celia 71 Makarova Tatiana 586 López García Zaira 614 Makasa Innocent 285 Lopez-Gonzalez Jose Maria 283, 317, 391 Maldonado-Uquillas Michelle Kelly 269, 497 Lopez-Gonzalez Paola 283, 317 Malewski Tadeusz 419, 494 López-Matayoshi César 415, 482 Malgosa Assumpció 326, 426 Lopez-Oceja Andrés 168 Malik Arif 70 López-Onaindia Diego 265 Malyarchuk A. Boris 23

736 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Manabe Sho 205, 583 Mayr R. Wolfgang 267 Man Chen 296 Mazzarelli Debora 123 Mao Jiong 689 McCarthy Loren 53 Marano Leonardo 633 Mccord Bruce 660 Marazita Mary 15 Mcgovern Catherine 610 Marca La Angelo 368, 418, 428, 455, 506 McGuigan John 493, 611 Marcińska Magdalena 403, 480 Mckiernan Heather 529, 581 Marcorin Letícia 635 McLaren Robert 458, 481, 571 Marcus Katrin 676 McLaughlin Patrick 522 Marimon Maria Jose 78 McMahon P. Timothy 63 Marini Chiara 122 McNevin Dennis 39, 84, 240, 304, 619, 650, 707 Marino Alberto 176, 406, 627–628 Meakin Georgina 243, 246 Marino Miguel 85, 385 Medellín Suárez Dayana 289 Marino Raffaella 122 Meijers Erin 711 Markovic Keckarevic Milica 292, 439 Melchionda Filomena 262, 448, 623, 687 Mármol Paula 415, 482 Melia Lisa 275 Marrubini Giorgio 123, 448 Meloni Valentina 525 Marshall Charla 63, 227, 431 Mendoza Libardo 284 Marsico Franco 369, 405 Meng Meng 332–333 Marti Matteo 102, 122 Mercer Claire 704 Martín-Arrebola Marta 415, 482 Mereacre Valeria 199 Martin Belinda 491, 496 Merino-Viteri Andrés 127 Martinez Beatriz 357 Metcalf Jessica 69 Martínez-Cortés Gabriela 318 Meurs B.J. van Joyce 663 Martínez Gustavo 310 Meyer Matthias 222 Martínez-Jarreta Begoña 305 Meyer Miriam 705 Martínez José Juan 310 Meyer Nele 166 Martinez Juliana 303, 324 Meyer Strunge Olivia 19, 639, 654 Martinez-Labarga Cristina 426 Miao Longfei 360 Martinez Lamper Luciano 342 Michalak Eliza 92, 371 Martínez Usaquén William 289 Michał Boroń 23 Martin Lorna 238 Mienkerd Sirirat 250 Martin Pablo 380 Miguel Mosquera Ana 21, 512, 615, 622, 624, 650, 662 Martins Silvia 162, 174, 193–194, 214, 321 Mihajlovic Milica 292, 439 Martos-Gago Veronica 675 Miladinov Takić Dijana 272, 309 Masato Funayama 103 Milan Jennifer 56 Mason-Buck Gabriella 121, 186, 414 Milić Aleksandra 503 Matsumoto Tomohiro 141 Miller W.P. Kevin 209 Matsushima Yutaka 649 Milnthorp Heather 529 Matzoll Ashleigh 56 Milot Emmanuel 397 Maurín Beatriz Gladys 310 Minaguchi Kiyoshi 377 Mautner E. Martin 449 Minawi Angelika 540 Mavrommatis Michalis 501 Minervino Costa Aline 342, 346 May Celia 109 Mingoia Marina 262 Mayers Carl 203 Ming Tianyue 155 May Julian 74 Minuti Barbara 438

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 737 Miozzo Cecilia María 310 Nam eun Da- 105 Miranda Araújo Daniel 342 Naqvi Sahin 15 Misas Florez Alison 276 Narváez-Narváez A. David 127 Mishel Stephenson-Ojea 271 Naue Jana 632 Mishra Aditi 315 Naughton Kristen 209 Misinzo Gerald 277 Navarro-Vera Isabel 595, 721 Mizuno Natsuko 143, 604 Nazir Muhammad 387 Modrow Jan-Hendrik 166 Nazir Shahid 323 Mogensen Smidt Helle 216, 364, 526 Neagu Elena 410 Mohammed Jaaved 15 Neckovic Ana 180 Molina Felipe Tula Marcos 310 Nedić Dalibor 311, 468, 503 Montes Núria 326 Neis Maximilian 114 Monticelli Fabio 239 Nelis Eva 479, 483 Montoya Zea Sara 289 Neocleous Tereza 592 Moon Hyeon Mi 513 Neri Margherita 102, 122 Moon Sang-ok 245 Neto Soares Moura Rodrigo 416 Moore David 88, 435, 562 Nettakul Anillada 250 Moore Tyiesha 534 Neubauer Jacqueline 199 Moraes V. Adriana 554 Neuhuber Franz 178 Moratelli Ricardo 174 Ng Hui Qi Eileen 411 Moreno Lilliana 498 Ng Kiat Boon 407, 411 Moretti Patrícia 140 Ng Shilen 136, 228 Morf Nadja 229, 242 Nguidi Masinda 321, 357 Morimoto Chie 205, 583 Nguyen Hung Duc 392 Morling Niels 216, 337, 364, 374, 388, 639 Nguyen Vivian 546 Moro Pedrosa Susana 76 Nicolae Alis Renata 410 Morroni Gianluca 262 Niederstätter Harald 14, 563, 612, 620 Mosavi Sayed 110 Nielsen Broman Ida 72 Mujika-Alustiza Antonio Jose 168 Niess Constanze 263 Mulero Julio 424, 436, 546 Nigam Kriti 341 Mulima Elias 277 Nijveld Alwart 26 Müller Eva 239 Nogel Monika 638, 702, 713–714 Müller Helena Linda 231 Noh Jeongsu 235 Muller Jonna 374 Nongmanee Wannarat 505 Müller Petra 286, 380 Nontiapirom Ketsaraporn 425 Muramatsu Hisanori 467, 695 Norona Wilma 424 Murphy Erin 36 Norton L. Heather 635 Musiał Jadwiga 133 Nothnagel Michael 624, 648 Muslin Claire 127 Nunes Larissa 140 Nagai Atsushi 336 Núñez Carolina 258 Nagel Jord 568 Núñez Fabián Naranjo Luis 127 Nagy Marion 394 Nunzio Di Aldo 401 Naji Mohammed 370 Nunzio Di Ciro 65, 144, 401 Nakahara Hiroaki 143 Nunzio Di Michele 65, 116, 144, 401 Nakanishi Hiroaki 143, 604 Nuss Niklas 166 Nakatome Masato 377 Nutini Lucia Anna 547

738 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Obal Marcel 132 Pancorbo M. De Marian 168, 236, 258, 297, 305, 320, 409, 489, Obayashi Masahiro 141 517 Occhi Gianluca 628 Panok Laksika 134 Ochiai Eriko 377, 649 Pantoja Jaime 710 O’Gorman Kieran 222 Panvisavas Nathinee 134, 425, 558 Oh Hyehyun 131 Papież Anna 630 Oh Na Yu 625 Parafiniuk Mirosław 251 Oh Sehee 130 Parker Glendon 56 Ohuchi Tsukasa 103, 636 Park Hee Sun 241 Okolie Victoria 328, 357 Park Hyun-Seung 131 Olasagasti Félix 236 Park Young Jee 131 Oldoni Fabio 30, 674, 688 Parra David 76 Olekšáková Tereza 76 Parra J. Esteban 635 Oliva Fernández Coro 706 Parsons Thomas 150, 208, 499, 532 Olivares Mogollón Fernanda 289 Parson Walther 61–63, 73, 191, 198, 229, 242, 286, 295, 348, Oliveira Luiza Guimarães de Maria 635 352, 374, 380, 390, 394, 502, 540, 563, 620, 624, 630, 650, 678 Oliveira Silviene 140 Parys-Proszek Agnieszka 403, 480 Olsen Gunn-Hege 287, 432 Patel Jayshree 53, 645 Patrick Basset 599 Olsen Jordyn 466 Patrizia Cormaci 368 Olsen Marita 287, 432 Patrizia Nespeca 708 Olson Sheri 650 Paz-Cruz Elius 269, 274 Omedei Monica 592 Paz-y-Miño César 273, 282, 319, 340, 356, 358 Onofri Valerio 187, 448, 623 Peck Michelle 150, 208, 472 Oorschot van Roland 46, 50, 80 Pedro Idigoras 78 Oorschot van Roland 108, 180, 219–220, 248 Peist Ralf 456, 492 Opperman Stephanie 580 Pelo Elisabetta 302, 438, 545, 547 Osawa Motoki 377, 649 Pelotti Susi 65, 151, 448 Ossowski Andrzej 163–164, 202, 212, 251, 255, 392, 528, 616 Penacino Belen Antonella 495 Otani Masanori 143 Penacino Gustavo 400, 495 Ou Xueling 204 Peng Dan 331, 381 Oven van Mannis 433 Peng Duo 55, 196, 233, 552, 555, 689 Owusu Loretta 466 Pepiński Witold 371, 494 Oyervide Maldonado Diana 273, 356 Pereira Filipe 76 Oz Carla 650 Pereira Luisa 507 Pääbo Svante 222 Pereira Luis Eburneo Alison 635 Pacholíková Petra 572 Pereira Rui 76 Pádár Zsolt 206, 638, 702, 713–714 Pereira Vania 170, 191, 337, 364, 388, 526 Paintner Carla 261 Perepechina Irina 363, 446, 591 Pajnič Zupanič Irena 119, 125, 132, 154, 179, 200, 234, 516, 521 Perez del Valle David 78 Pakulla-Dickel Sarah 492 Pérez-M Gabriela 282 Palacio Oscar 497 Pérez-Villa Andy 273, 282, 319, 340, 356, 358 Palchetti Simona 438 Perry H. George 499 Palencia-Madrid Leire 258, 297, 489 Pesaresi Mauro 187, 687 Palomo-Díez Sara 96, 138, 185, 265, 301, 326, 415, 482 Peters Annette 663 Pamjav Horolma 270 Peters Elizabeth 650

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 739 Petersen Bie Cathrine 526 Preuß-Wössner Johanna 99 Petersen Frederik Johan 526 Previderè Carlo 42, 123, 448, 516, 718 Petersen Leif 74 Prieto-Fernández Endika 236 Petković Stojan 468, 503 Prieto Lourdes 25, 686 Pfeiffer Heidi 244, 247, 608, 621, 659 Primignani Paola 406 Pflugbeil Anne-Marie 582 Prinz Mechthild 522, 536 Phetpeng Sukanya 505 Prochnow Anke 456, 492, 613 Phillips Christopher 14, 21–22, 36, 61, 208, 229, 304, 388, 512, Proff Carsten 459 569, 614–615, 622, 624, 644, 650, 662, 675, 677–678 Prosser Chris 611 Phua Ho Cheng 542 Pruiett Caron 261 Piccinini Andrea 167, 406, 718 Puac Fedja 292, 439 Pico Adriana 338 Puente de la María 14, 512, 612, 620, 624, 650, 678 Picornell Antonia 278, 290 Pugh Simone 610 Pic-Taylor Aline 140 Pu Yan 87, 420 Pietroni Carlotta 74 Pydi Sudheer Satya 217 Pilli Elena 86, 556 Qian Jialin 698 Piña-Dzul Adriana Karla 283, 317 Qian Xiaoqin 374 Piñero Herrera Mariana 369, 405 Qiao Lu 360 Pinheiro Marcelo Luiz 321 Qiao Xianhua 197 Pinto Nádia 225, 400, 584, 601, 609 Qifan Sun 89, 576 Piotrowska-Cyplik Agnieszka 104, 157, 169, 189 Qingfeng Chen 93 Pirro Hysi 648 Que Tingzhi 332–333 Pisarek Aleksandra 620, 630, 644 Qu Shengqiu 299, 398, 573, 669, 689, 692 Pistono Alice 243 Raats Nathalie 716 Pittner Stefan 239 Rabbach Dawn 514 Płoski Rafał 16, 20, 23 Rabitti Luciana 369, 405 Podini Daniele 30, 674, 688 Raczkowski Michał 133 Podovšovnik Eva 119, 179, 234 Radanovic Anja 439 Pogorelc Gornjak Barbara 132, 179, 200, 516, 521 Raddi Sara 404 Polańska Joanna 630 Radjabzadeh Djawad 71 Polizzi Nicolò 42 Radojicic Verica 292, 439 Polverari Silva Fernanda 279–280, 303, 324–325 Rådström Peter 709 Ponzano Elena 65 Rafati Alireza 656 Poór Viktor 206 Raffone Caterina 258, 265, 326, 409, 489 Porto João Maria 76, 106, 147, 237, 314, 349, 386, 389, 488, 607 Raimrez Andres Carrasco Jose 463 Porto Marika 628, 677 Rajagopal Swetha 499 Posada Yeny 276 Rakha Allah 323 Pośpiech Ewelina 16, 20–21, 23, 615–616, 620, 630, 644, 648, Ralf Arwin 27, 71, 433 653 Rambo Taylor 611 Potabattula Ramya 630 Ramella Isabel María 310 Power Daniel 491, 650 Ramesar Raj 238 Powierska-Czarny Jolanta 104, 133, 157, 169, 189, 288 Ramon Cori 278, 290 Pozo Alejandra Ruiz Viviana 471 Rangel-Villalobos Hector 271, 283, 317–318 Pradujkanchana Jintana 443 Ranran Li 576 Presciuttini Silvano 42, 123, 401 Rasal Raquel 326 Press Rich 712 Raul Jean-Sébastien 211

740 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Raymond Jennifer 17, 619 Roman G. Madeline 116, 144 Real Corte Francisco 237, 314, 349, 386, 607 Romanini Carola 111 Rębała Krzysztof 23 Romero Magdalena 43, 101, 111 Reem Almheiri 685 Romsos Erica 473 Refn Mie 337 Roocke Sascha 676 Re He 268 Roosenboom Jasmien 15 Reither Jack 108, 248 Rosell-Herrera Rocío 415, 482 Ren He 160, 296 Roseth Arne 47 Rennie Jarrad 442, 524 Rosli Razanah Afiqah 407, 411 Rensfield Anthony 515 Roth Chantal 198, 557, 563 Rerkamnuaychoke Budsaba 570 Rothschild A. Markus 114, 652 Revoir Andrew 504 Rotondo Martina 101, 111 Reyes Patricio 710 Rowe Emily 32 Ribeiro Maximo C. Talita 532 Rožić Sara 260, 352 Riccardi Natalia Laura 421 Rubi-Castellanos Rodrigo 283 Ricciardelli Rossana 718 Ruiz-Ramirez Jorge 512 Ricci Pietrantonio 401 Russo Daniel 342 Ricci Ugo 302, 438, 545, 547 Russo Giancarlo 79, 199 Rice Robert 56 Rutherford Donna 37 Richards Rebecca 645 Ryan Suzanna 243, 246 Richmond Stephen 15 Sabadra Priti 129, 152 Rickards Olga 634 Sabahanaloh Nuryanee 505 Rihova Pavla 112, 252, 291 Sabbagh Audrey 68 Riman Sarah 33–34, 257 Sadam Maarja 487, 500 Ring Joseph 63, 227 Saia Flavio 401 Ristow Gustav Peter 354 Saidi Laiq-Jan 231 Rivero-Salazar Rocio Maria 317 Saito Kazuyuki 143, 604 Robertson James 39, 707 Sajib AA 650 Roberts Richard 222 Sala Andrea 43, 359, 535 Robino Carlo 65, 151, 404, 448 Salinas Viviana 710 Roca Gabriela 466 Salvo Mjølsnes Nina 287 Rocandio María Ana 305 Salvoro Cecilia 628 Rocca Della Chiara 350, 575, 592 Salzmann Andrea 79, 195 Rocchi Anna 65 Samie Lydie 172, 577 Rocha Andrea 101 Sampaio Lisa 76, 106, 389, 488 Rocha Natalia Andrea 111 Sánchez Adrián Luis 310 Rockenbauer Eszter 170 Sanchez Izquel 284 Rodrigues Diogo 386 Sanchez-Pla Alex 585 Rodríguez Cristina 322, 335, 340, 402 Sánchez-Polo Alejándra 185 Roewer Lutz 374, 394, 540 Sanga Malin 544 Rogalla-Ładniak Urszula 23 Sangha Jangbir 110 Roh Jee Ye 186 Sangpueng Sireethron 396 Rohleder Udo 229, 242 Sano Kaito 679 Rolf Burkhard 441 Sanqoor Shaikha 387 Rolo Mariana 106 Santos Cristina 326, 426 Romagné Frédéric 222 Sarik Heather 209

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 741 Sarman Andrew 53, 382, 417 Shapturenko Marina 641 Sarno Stefania 65 Sharavdorj Purevdulam 393 Sato Shiori 679 Shaw Jeff 571 Saw Ling Yih 529 Sheehan Nuala 441 Scarnicci Francesca 65, 151 Sheng Hu 89, 576 Schaapveld Tayla 580 Shen Xuefeng 437, 593 Scheer van der Mikle 711 Shen Yiwen 52 Scheiper Stefanie 263 Shi Chunfeng 264 Schelb Márcia 554 Shih Shelly 427, 457, 538 Schellberg Tim 344 Shim Hyeonah 131 Scherer Mario 456, 492 Shinkevich Marina 641 Scherer Zanenga Luciane 532 Shin Kyoung-Jin 379, 513 Schleenbecker Uwe 229, 242 Shin Woung Hyun 253 Schmidt Max 247 Short Marc 198, 436, 546 Schmitt Cornelia 114 Shrivastava Divya 341, 685 Schnaller Lisa 563 Shrivastava Pankaj 315, 341, 530, 682, 685 Schneider Peter 36, 114, 263, 531, 540, 646, 650, 652 Shriver D. Mark 15 Schöbel Kerstin 114 Shunkov Michael 222 Schönborn Holger 582 Shyla Alena 256 Schroeder Scott 129 Sibilla Gustavo 310 Schuerenkamp Marianne 621 Sibte Hadi 90–91, 175, 383, 670–671 Schultheiss Eva 137, 540 Siciliano Salvatore 214 Schulz Iris 120, 215, 701 Sidstedt Maja 617, 655, 709 Schury Natalie 459 Siemens Helena 453 Schwark Thorsten 166 Sijen Titia 26, 412, 551, 590, 603, 673, 686, 711, 717 Schwender Kristina 621, 659 Sikora-Polaczek Marta 630, 644 Scott Kirstie 70 Silingardi Enrico 145 Scudder Nathan 39, 707 Silva Alves de Jesus Flávia 280, 303, 324 Sears Alison 17, 619 Silva Beatriz Candelária da Amanda 635 Séguin Diane 703 Silva Benedita 681 Sekiguchi Kazumasa 143 Silva Dayse 162, 174, 193–194, 214 Sell Christian 540 Silva do Valle Guilherme 635 Semikhodskii Andrei 586 Silva Maya W. Isabela 532 Sensabaugh George 457 Silva Rosane 416 Senst Alina 701 Silva Vieira Da Cláudia 237, 314, 349, 386, 607 Seong Min Ki 130 Silvery Janine 453 Serin Ayse 182, 343, 367 Simão Filipa 274, 321, 353, 357, 390 Serra Armando 106, 389, 488 Simkova Halina 683 Serventi Patrizia 176, 627 Šimková Halina 24 Severini Simona 691 Simões Luiz Aguinaldo 635 Sevillano Rubén 236 Simone Di Paola 42 Sguazzi Giulia 509 Simón Fernández de Lourdes 706 Shaffer R. John 15 Siqueira Emilia E. Maria 554 Shan Adnan Muhammad 337 Sjölund Peter 40 Shan Shaodong 360 Skawrońska Małgorzata 371 Shao Chengchen 52, 153, 574 Skillman Jessica 472

742 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Slavkovský Rastislav 618, 631 Stock April 664 Slon Viviane 222 Stojiljković Goran 311 Slooten Klaas 31, 606 Storts Douglas 458, 481, 514, 571 Smith Rebecca 121, 186 Story Joanna 373 Smit Sophie 686 Stránská Jana 618, 631 Snipen Lars 79 Stravers Lydia 673 Snyder-Leiby Teresa 493, 611 Streba Irina 177 Sojikul Punchapat 425 Strnad Zdenek 291 Soliven NA 650 Strobl Christina 198, 295, 563 Soltyszewski Ireneusz 419, 494 Sturk-Andreaggi Kimberly 63 Sołtyszewski Ireneusz 371 Suarez Philippe 640 Song Feng 165, 451, 485, 690, 696 Subhani Zuhaib 88 Song Mengyuan 597 Subirà Eulàlia M. 326 Song Wei 210 Subramanian Aishwaryaa 674 Song Yutong 366 Su Chih-Wen 232 Sorçaburu-Ciglieri Solange 448, 516 Sugano Yukiko 467, 695 Šorgić Dejan 272, 309 Sukser Viktorija 260, 352 Sorror Fatma 375 Sundaram Sudha 22 Sosa-Escalante Enrique Javier 283, 317, 391 Sun Hongyu 331, 381, 437, 593 Sousa Antão Sofia 601, 609 Sun Kuan 330, 574 Sousa José Carneiro de Maria 106 Sun Qifan 124 Speed Belinda 511 Suren Ganbold 393 Spiden Michelle 664 Svidizinski E. Arthur 554 Spólnicka Magdalena 16, 20, 23, 616, 653 Syn Kiu Choong Christopher 136, 223, 228, 407, 411, 637, 647 Spradley Kate 164 Szargut Maria 163–164, 202, 212, 251, 255, 392, 616 Sprecher Cynthia 514 Szkuta Bianca 108, 180, 243, 248 Springer Eliot 522 Szűcs Dominika 206 Srisiri Korapin 570 Tacha Hicks-Champod 599–600 Srivastava Ankit 341 Tagliabracci Adriano 187, 262, 448, 623, 687 Staadig Adam 617, 655, 680 Taira Ryan 486 Stacey Janet 275, 382, 417 Takada Aya 143, 604 Staiti Nicola 176, 406, 556 Talarico Anna 102 Starke Anton 328 Tamaki Keiji 205, 583 Staufer Christian 239 Tanasic Vanja 292, 439 Štefanová Helena 618, 631 Tang Qiqun 52, 153, 574 Stefanović Aleksandra 272, 309 Taniguchi Kei 224 Steffen Becky 226 Tan Jiayu 136, 228 Stein Christina 629 Tanković Dijana 532 Stenzl Vlastimil 307 Tan Ying Ying Jolena 637, 647 Stephens Kathryn 374, 472, 515 Tan Yu 55, 196, 233, 300, 579, 669, 672, 692 Stepien Jennifer 676 Tao Ruiyang 95, 210, 329, 360, 696 Stevanović Milena 23 Taroni Franco 45, 67, 577 Stevenson Kate 645 Tartera Enric 326 Sticchi Maurizio 406 Tasa Gunnar 487, 500 Štikarová Radka 142, 293 Tauhyl Paula Ana 532 Stobl Christina 191 Tavallaei Mahmoud 656

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 743 Tay Jacquelyn 407, 411 Umino Keishi 467, 695 Taylor Cassandra 63 Ungria Corazon A De Maria 650 Taylor Cassie 227 Usui Kiyotaka 103, 636 Taylor Duncan 32, 48, 423, 566, 578 Utz Silvia 510 Taylor Melissa 712 Vaca Mishelle 402 Tebbe Frederike 137 Vai Stefania 86 Teodoridis Jens 540 Vakula Svetlana 641 Teul Iwona 212 Väli Marika 487, 500 Teumer Alexander 663 Vallone Peter 33–34, 226–227, 257, 473, 712, 720 Thakur Nisha 285 Vándor Anna 270 Thanakiatkrai Phuvadol 173, 443, 505, 542 Vanek Daniel 112, 161, 201, 252, 291 Thompson Jonelle 458, 481 Vangbo Mattias 484 Thomson Jim 37, 559, 562 Vankova Lenka 252 Thong Zhonghui 637, 647 Vapenik Lukas 572 Thore Egeland 589 Vargas Clara 338 Tian Huan 55, 196, 233, 552, 555, 669 Vašek Jakub 142, 293 Tiemann Carsten 453 Vasiljević Perica 272 Tikalova Eva 112, 252 Vasut Radim 307 Tillmar Andreas 38, 40, 75, 208, 362, 588, 596, 617, 655, 680 Vazza Giovanni 628 Tim Clayton 562 Vecchio Simona 145 Tineo Herman Dean 390 Vegas Esteban 585 Tizzano Eduardo 326 Vejl Pavel 142, 293 Todd James 493, 611 Vela Margarita 322, 335, 340, 402 Tokunaga Katsushi 679 Velásco-Vázquez Javier 185 Tomasek Petr 201 Vella Di Giancarlo 404 Tommolini Federica 691 Vella Joanna 394 Toogood Hayley 246 Veltre Virginia 634 Toscano Adelina Michelle 269 Vennemann Marielle 244, 247, 608, 621, 659 Tozzo Pamela 151, 708, 718 Verhoff A. Marcel 263 Trajkovska Ljubica 384 Verstraete Paulien 64 Trimborn Marc 540 Verzeletti Andrea 65, 151, 421, 565, 667, 718 Trinks Julieta 347 Vidaki Athina 71, 657, 663, 666 Trombetta Beniamino 350 Vigeland Dehli Magnus 589 Troup Charles 484 Vignali Giulia 167 Truelsen Ditte 388 Vijayan Ranjit 387 Tuitoga Naomi 650 Vijaychander Sharada 281, 557 Turchi Chiara 65, 187, 448, 623, 687 Villaescusa Patricia 297, 305 Turner Benjamin 488 Vilsen Byg Søren 216 Turrina Stefania 395, 440 Vincent Castella 599 Tu Zheng 183, 422 Vincenti Marco 575 Tvedebrink Torben 59, 216, 364, 594, 602 Viset Auttapol 505 Ueland Maiken 84, 240 Visser de Leroy 27 Uerlings Sonja 83 Viviana Pugin Ojeda 463 Uitterlinden G. André 71, 663 Volk Paris 139 Ujchariyakajorn Arjaree 505 Voss Arévalo Cristina 76 Ulubay Ayca 367 Votrubova Jitka 112, 161, 201, 252

744 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Vranes Miroslav 456, 492 Welti Susan 472 Vreede de Jennifer 26 Wepeba Pet-Paul 376 Vuković Radenko 468 Werner Michaela 413 Vullo Carlos 43, 101, 111, 328, 357 Westring Christian 413 Wagner Samantha 711 Wetton Jon 109, 188, 213, 351, 373 Währer Jonathan 120 Wheaton Ariana 152 Waitara Leticia 277 White D. Julie 15 Waiyawuth Worawee 250, 396 Wiegand Peter 207, 247, 453, 527, 540 Waldenberger Melanie 663 Williams Graham 146, 254, 447, 475, 478 Walichiewicz Paulina 515 Willis-Barrett Skye 414 Walsh J Simon 39, 707 Willis Sheila 712 Walsh Susan 15, 648 Willuweit Sascha 374, 540 Walton-Williams Laura 254, 447 Wilson Roy 663 Wang Bao-jie 97, 339 Wilson-Wilde Linzi 44 Wang Chong 422 Wisner Mary 538 Wang Hongran 332 Wit de Denise 28 Wang Hui 552, 555, 689 Wójcik Monika 371 Wang Jiaqi 408 Wolff de Tialda 80 Wang Junyan 117 Wolf Kathrin 492 Wang Le 124, 361 Wolford Nicholas 581 Wang Li 300, 579, 669, 672, 692 Wongvoravivat Chalampoo 396 Wang Mengchun 454 Wong Yee Hang 136 Wang Mengge 312, 378 Wong Yongxun 407, 411 Wang Nana 331, 381, 593 Wood Zoe 203 Wang Ning 519, 561 Wootton Sharon 30, 281, 433, 557, 563, 650, 674 Wang Qian 55, 117, 233, 548, 661, 665 Woźniak Anna 16, 20, 23 Wang Qiuyue 126, 469, 533 Woźniak Marcin 23 Wang Shouyu 696 Wróbel Maria 403, 480, 528 Wang Shuangshuang 165, 451, 485 Wu Huijuan 698 Wang Tzuming 183 Wu Jianqiang 519, 561 Wang Xudong 98 Wu Riga 331, 381, 437 Wang Yan 454 Wurmb-Schwark von Nicole 166 Wang Yanyun 165, 233, 451, 665 Wu Xue 97 Wang Yicong 204, 519, 523, 561 Wysocka Joanna 15 Wang Yinji 299, 573, 689, 692 Xavier Catarina 14, 390, 502, 612, 620, 630, 650, 678 Wang Yongqing 669 Xiao Xiao 55, 233 Wang Yufang 126, 469, 533 Xia Xi 97, 339 Wang Zheng 155, 197, 312–313, 334, 378, 597, 690, 696 Xie Bowen 165, 451, 485 Wan Ning 611 Xie Jianhui 52, 153, 574 Watanabe Ken 224 Xie Mingkun 485 Watsula Dan 110, 209 Xie Yifan 204, 519, 523, 561 Wee Xue Ming 223 Xing Jia-xin 97, 339 Weinberg M. Seth 15 Xuan Jin-feng 97, 339 Weirich Volker 445 Xu Feng-ling 97 Weitz Steven 534 Xu Qiannan 100, 474, 700 Weksler Marcelo 214 Xu Xuewei 360

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 745 Xu Zhen 183 Zhang Chi 148, 361 Yadamsuren Oyunchuluun 393 Zhang Chuanqi 665 Yadav Kumar Vijay 341 Zhang Gengqian 298, 408, 454 Yamamoto Toshimichi 336, 679 Zhang Ji 126, 469, 533 Yang Guifang 204, 519, 523, 561 Zhang Jiashuo 210, 360 Yang Jiawen 87, 420 Zhang Jiayi 100, 474, 700 Yang Lin 523, 561 Zhang Jingjing 18, 82 Yang Qinrui 52, 153, 574 Zhang Jingyi 95, 210, 329, 360 Yang Tae-Jin 131 Zhang Jjiashuo 95 Yang Yiwen 126, 469, 533 Zhang Lin 55, 87, 196, 233, 299–300, 398, 420, 552, 555, 573, Yang Zihao 95, 329 579, 661, 665, 669, 672, 689, 692 Yan Jiangwei 18, 82, 268, 296, 298, 408, 454, 698 Zhang Linan 366 Yan Shi 160 Zhang Ranran 300, 669, 672, 692 Yao Jun 97, 339 Zhang Suhua 95, 210, 329, 332, 360 Yao Liu 89 Zhang Yanyan 204, 519, 523, 561 Yao Yining 574 Zhang Yilun 332–333 Yepez-Santos Ignacio José 274 Zhang Yinming 437, 593 Ye Yi 690 Zhang Zhang Ranran 579 Ye Ziwei 334 Zhao Chenxi 597 Yi Hai 264 Zhao Huan 485 Yin Lu 552, 555, 573, 689, 692 Zhao Jing 18, 268, 296, 454, 698 Yixia Zhao 89, 576 Zhao Yixia 124 Yizengaw Ajanaw 404 Zhao Zhenmin 100, 474 Y.Koh Lin 94 Zhao Ziqin 52 Yoneyama Katsumi 143 Zheng Mingrui 155 Yoon Leena 674 Zheng Yu 523 Yoshimoto Takashi 679 Zhihan Zhou 330 Yoshimura Sumitaka 141 Zhong Chang 424 Young Brian 249 Zhou Chengjiang 332 Young Jennifer 70, 491 Zhou Yanping 155 Yu Jinwen 561 Zhou Yijun 126, 469, 533 Yumiceba Verónica 273, 282, 319, 340, 356, 358 Zhou Yuxiang 52, 574 Yun Keming 298, 408 Zhu Hong 197, 313, 334, 690 Yu Zailiang 264 Zhu Jing 299, 398, 573, 689, 692 Żaba Czesław 92, 371 Zhu Qiang 126, 469, 533 Zahrer Waltraud 178, 239 Zhu Ruxin 366 Zambrano Karina Ana 273, 282, 319, 322, 335, 340, 356, 358, Zieger Martin 510 402 Zielinska Grażyna 392 Zavala Elena 222, 499 Zielińska Grażyna 163–164, 202, 212, 251, 255, 616 Zavarin Vladislav 586 Zieliński Piotr 20 Zayerka Yulia 256 Zimmermann Bettina 352, 394, 563 Zbieć-Piekarska Renata 23 Zinkova Alzbeta 683 Zehner Richard 643 Zin Thant 558 Zeinali Sirous 294 Ziqin Zhao 330 Željana Bašić 499 Zook Justin 720 Zgonjanin Dragana 90–91, 311, 468, 503 Zoufal Peter 612

746 THE 28TH CONGRESS OF THE INTERNATIONAL SOCIETY FOR FORENSIC GENETICS Zou Xing 313 Bios Consortium 663 Zubańska Magdalena 16, 20, 23, 653 on behalf of Gefi Group 537 Zucchelli Luca 628 on behalf of the VISAGE Consortium 630 Zuidberg Matthijs 80 The CaDNAP Group 229, 242 Zuniga Sofia 26, 551 The dna.bases Consortium 62, 348 Zupanc Tomaž 132, 179 The DNASEQEX Consortium 286 Zvènigorosky Vincent 68, 211 The VISAGE Consortium 624 Visage Consortium 648 Walther Parson on behalf of the VISAGE Consortium 14, 612

9 – 13TH SEPTEMBER 2019, CZECH REPUBLIC, PRAGUE CONGRESS CENTRE 747