Nova Southeastern University NSUWorks
Biology Faculty Articles Department of Biological Sciences
8-14-2018 Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments Maria A. Nieves-Colon Arizona State University
Andrew T. Ozga Arizona State University, [email protected]
William J. Pestle University of Miami
Andrea Cucina Universidad Autonoma de Yucatan
Vera Tiesler Universidad Autonoma de Yucatan
SeFoe nelloxtw pa thige fors aaddndition addal aitutionhorsal works at: https://nsuworks.nova.edu/cnso_bio_facarticles Part of the Biological and Physical Anthropology Commons, and the Biology Commons
This Article has supplementary content. View the full record on NSUWorks here: https://nsuworks.nova.edu/cnso_bio_facarticles/972
NSUWorks Citation Nieves-Colon, Maria A.; Andrew T. Ozga; William J. Pestle; Andrea Cucina; Vera Tiesler; Travis W. Stanton; and Anne C. Stone. 2018. "Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments." American Journal of Physical Anthropology 166, (): 824-836. doi:10.1002/ajpa.23472.
This Article is brought to you for free and open access by the Department of Biological Sciences at NSUWorks. It has been accepted for inclusion in Biology Faculty Articles by an authorized administrator of NSUWorks. For more information, please contact [email protected]. Authors Maria A. Nieves-Colon, Andrew T. Ozga, William J. Pestle, Andrea Cucina, Vera Tiesler, Travis W. Stanton, and Anne C. Stone
This article is available at NSUWorks: https://nsuworks.nova.edu/cnso_bio_facarticles/972
Page 1of24
between this versionandtheVersionofrecord. Pleasecitethis articleasdoi:10.1002/ajpa.23472. through thecopyediting, typesetting, paginationandproofreadingprocess, whichmayleadtodifferences This istheauthormanuscript acceptedforpublicationandhasundergone full peerreviewbuthasnotbeen BCS 0612727, the Rust Family Foundation, the Leakey Foundation, Arizona State University University State Arizona Foundation, Leakey the Foundation, Family Rust the BCS 0612727, Strategic Initiative Funds, Fundación Roberto Hernández, Fundación Pedro y Hernández, Elena Pedro Fundación Hernández, Fundación Roberto Initiative Funds, Strategic Funding for this work was provided by National Science Foundation awards BCS 1622479 and BCS 1622479 awards Foundation ScienceNational by provided work was forFunding this Grant Support: and Jerry Foundation, Murdoch. Selz the Maria A. Nieves Colón list: Author environments Ancient DNA; Tropics; DNA extraction; Skeletal remains; Next Generation Sequencing; Sequencing; Next Generation remains; Skeletal DNA extraction; Tropics; DNA; Ancient Keywords: protocols extraction of ancient DNA Comparison Title: Abbreviated tables: of Number bibliography: text plus pages of Number Tempe, AZ 85287 2402 872402 Box P.O. University State Arizona Change and Social Evolution of Human School Maria A. Nieves Colón author: Corresponding [email protected] 1 from tropical for remains skeletal protocols DNA extraction ancient of two Comparison Title: 7 6 9700 México 5 4 3 2 Number of figures: 4 figures: of Number Number of supplementary files: files: supplementary of Number
Tiesler School of Human Evolution and Social Change, Arizona State University, Tempe, AZ 85287 Tempe, AZ 85287 University, State Arizona Change, and Social Evolution of Human School Institute of Human Origins, Arizona State University, Tempe, AZ 85287 University, State Tempe, AZ 85287 Arizona Institute Origins, of Human Center for Bioarchaeological Research, Arizona State University, Tempe, AZ 85287 AZ 85287 University,State Tempe, Arizona Research, Center for Bioarchaeological 92521 CA Riverside, Riverside, of California University Department of Anthropology, Mérida, Yucatán, de Yucatán, Autónoma Universidad Facultad Antropológicas de Ciencias Gables, Coral FL 33124 of Miami, University Department of Anthropology, 85287 Tempe, AZ University, State and Arizona Medicine, Center for Evolution
5 Author Manuscript Stanton , Travis W.
2 2
1*
, Andrew T. Ozga This article isprotected by copyright. All rights reserved. 6 , Anne C. Stone , Anne C. American JournalofPhysicalAnthropology 3 3
1,2,3 16 1,2,3,7
, William J. Pestle , William J.
4 , Andrea Cucina , Andrea 5 , Vera
protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from fragments from DNA ultrashort recovery of for previously designed D, was Method protocol, skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an initial adding by the first H, modifies Method second, The remains. skeletal Results: step. and decalcification digestion an extended Materials and Methods: Materials and sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction extraction first Illumina The 2500. HiSeq the on shotgun sequenced and libraries, sequencing tropical sites in Tanzania, Mexico, and Puerto Rico (N=12). (N=12). Rico and Puerto Mexico, Tanzania, in sites tropical methods on ancient samples of teeth and petrous portions excavated from tropical and semi from tropical excavated portions of and teeth petrous ancient samples on methods results suggest that Method D is the optimal choice for working with samples from warm and from warm samples with choice for optimal working the D is suggest Method results that humid environments. Additional optimization of extraction conditions and further testing of and further testing conditions of extraction optimization Additional environments. humid far by poor ancient DNA yields. Here we compare the performance of two DNA extraction DNA extraction two the performance of yields. compare we Here ancient DNA far poor by surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our extremely is our fragmented, tropical contexts from remains or historic ancient surviving DNA in Method H with different types of samples may allow for improvement of this protocol in the the in protocol of this for improvement allow may types different of samples H with Method Discussion: Objectives:
shorter average DNA fragments. fragments. DNA shorter average type. However, Method H samples had higher endogenou had higher H samples Method type. However,
quality filtering, but also higher clonality. In con clonality.higher also but quality filtering, damage patterns recovered from samples extracted wit extracted from samples recovered damage patterns human habitation. However, paleogenomics research in these climates has been constrained so so been constrained has these climates in research paleogenomics However, habitation. human future. No significant difference was found in overall ancie overall in found was difference significant No The tropics harbor a large part of the world’s biodiversity and have a long history of have history a long and biodiversity world’s of the harbor part a large tropics The Author Manuscript But, since DNA. ancient endogenous recovered successfully Both methods This article isprotected by copyright. All rights reserved. All samples were extracted twice, built into double stranded into twice, built extracted were samples All American JournalofPhysicalAnthropology John Wiley&Sons, Inc. ABSTRACT ABSTRACT 1 trast, samples extracted with Method D had Method with extracted samples trast, h either method, irrespective of tissue of tissue irrespective eitherh method, s content and more mapped reads afterreads mapped and more content s nt DNA yields or post mortem yieldsor post mortem DNA nt Page 2of24
Page 3of24
proposed for improving endogenous aDNA recovery. Comparing different extraction methods, methods, extraction different recovery. Comparing aDNA endogenous forproposed improving by adding an initial EDTA wash, as in Warinner et al. (2014), and an extended digestion and digestion extended al. (2014), and an et EDTA Warinner wash, as in an initial by adding Nevertheless, despite the challenges of working with poorly preserved samples, several studies several studies samples, poorly preserved with working challenges of the despite Nevertheless, portion samples from degraded skeletal remains recovered from tropical sites in Tanzania, Tanzania, in sites from tropical recovered remains skeletal from degraded samples portion preservation is negatively correlated with thermal age due to the accelerating effect of high of high accelerating effect age the due to thermal with correlated negatively is preservation DNA because geographic focus its in be constrained to continues research paleogenomics Pääbo, 1989). Due to the variety of taphonomic and diagenetic processes that take place after after take place that diagenetic processes and variety of taphonomic the Due 1989). Pääbo, to which is highly susceptible to external contamination (Gilbert et al., 2006; Hofreiter et al., 2001; et al., 2001; Hofreiter et al., (Gilbert 2006; contamination external to highly susceptible is which Meyer et al., 2014; Orlando et al., 2013). et al., 2013). Orlando al., 2014; Meyer et dating as far back in time as the early Holocene and Middle Pleistocene (Meyer et al., 2016; al., 2016; et (Meyer Pleistocene Middle and Holocene as early the time far in dating back as
generation sequencing methods now allow the recovery of complete genomes from remains from remains genomes of complete recovery the allow now methods generation sequencing recovery of extremely short DNA fragments (as small as 30 base pairs) in ancient bone and tooth and tooth ancient bone base as 30 pairs) in (as small DNA fragments short recovery of extremely DNA (Ho & Gilbert, 2010). Recent advances in DNA extraction, target enrichment, and next target enrichment, DNA extraction, in Recent advances & 2010). DNA Gilbert, (Ho and South East Asia (Damgaard et al., 2015; Gamba et al., 2014; Gutierrez Garcia et al., 2014; et al., 2014; Gutierrez Garcia et al., 2014; Gamba et al., 2015; (Damgaard Asia and South East decalcification step as in Gamba et al. (2016). Method D was specifically designed to increase increase to designed D was specifically Method Gamba et al. (2016). as in step decalcification et al., 2015; Heintzman et al., 2015; Kehlmaier et al., 2017; Seguin Orlando et al., 2014). et al., 2014). Seguin Orlando al., 2017; Kehlmaier et et al., 2015; Heintzman et al., 2015; fragments of the autosomal genome or, alternatively, on multicopy loci such as mitochondrial as mitochondrial such loci multicopy on or, genome alternatively, autosomal fragments of the have successfully recovered aDNA from tropical sites in the Caribbean, the Yucatan peninsula peninsula Yucatan Caribbean, the the in sites from tropical aDNA recovered have successfully al., 2013a), hereafter Method D, to a second approach, Method H. Method H modifies the former the H modifies H. Method Method approach, a D, second to Method al., 2013a), hereafter recently dated human and animal remains, including at least one from a tropical context (Günther context a tropical one from including remains, at least animal and human recently dated 2013a). Because of this, until recently, aDNA studies have focused on short but informative informative but short on have focused recently,aDNA studies until of this, 2013a). Because regions, which have the highest chance of DNA survival (Paijmans et al., 2013; Wade, 2015). 2015). Wade, 2013; et al., (Paijmans DNA survival highest chance of the have which regions, Mexico and Puerto Rico (N=12). Specifically, we compare the method developed by (Dabney et (Dabney by developed compare we method the (N=12). Rico Specifically, and Puerto Mexico hominin fossils (Meyer et al., 2016; Meyer et al., 2014), and from a large variety ofa more large variety and from 2014), Meyer et al., al., 2016; (Meyeret fossils hominin in small, degraded DNA fragments (Allentoft et al., 2012; Briggs et al., 2007; Dabney et al., Briggs et al., 2007; 2012; et al., fragments (Allentoft degraded DNA small, in aDNA studies focus on archaeological remains excavated from cold and temperate world world and temperate from cold excavated remains archaeological on focus aDNA studies aDNA from Late Pleistocene cave bear remains (Dabney et al., 2013a), from Middle Pleistocene Pleistocene Middle al., 2013a), from et (Dabney remains caveLate bear Pleistocene aDNA from et al., 2001). Consequently, most genetic information obtained from ancient samples is contained contained is samples from ancient obtained genetic information most Consequently, et al., 2001). Hofreiter et al., 2015; Kistler et al., 2017; Lindahl, 1993; Smith et al., 2001). Therefore, most Lindahl, Smith 1993; et al., 2017; Kistler et al., 2015; Hofreiter conservation genetics, among other fields. fields. other genetics, among conservation (instead of guanidine thiocyanate). Method D has been successfully employed in the recovery of the in employed been successfully D has Method guanidine thiocyanate). of(instead death, DNA decays exponentially once cell repair functions cease in biological tissues (Hofreiter (Hofreiter tissues biological cease in functions cell repair once exponentially death, DNA decays temperatures on biomolecule decay and fragmentation (Adler et al., 2011; Allentoft et al., 2012; et al., 2012; Allentoft al., 2011; (Adler et and fragmentation decay biomolecule on temperatures recovery from these contexts is of great interest to advance anthropology, paleontology, and paleontology, of great anthropology, advance to is interest contexts recovery from these approaches in its use of silica spin columns and a guanidine hydrochloride binding buffer buffer binding guanidine and a hydrochloride columns spin use of silica its in approaches excavated from these environments and optimizing or improving methods that facilitate aDNA facilitate aDNA that methods or and improving optimizing environments from these excavated guanidium based salt to bind DNA fragments and remove inhibitors. It differs from previous It from previous remove differs inhibitors. and DNA fragments bind to salt guanidium based 2014; Buzas et al., 2002). Therefore, understanding how DNA is preserved in degraded remains remains degradedin preserved is DNA how Therefore, understanding et al., 2002). Buzas 2014; method employs a 24 hour proteinase K digestion to break up cell proteins, and uses a chaotropic a and uses chaotropic proteins, break to cell up K digestion proteinase employs a 24 hour method the tropics harbor a large portion of the world’s biodiversity and human settlements (Brown, settlements and human of biodiversity world’s the portion a large harbor tropics the extractions. In line with earlier protocols (Höss & Pääbo, 1993; Rohland & Hofreiter, 2007), this this Hofreiter, & 2007), Rohland 1993; Pääbo, &In (Höss protocols earlier with line extractions. Kehlmaier et al., 2017; Mendisco et al., 2015; Schroeder et al., 2015). Both today and in the past, past, the and in today Both et al., 2015). Schroeder et al., 2015; Mendisco al., 2017; Kehlmaier et Despite these improvements in stretching the time depth for aDNA recovery, recovery, depth for aDNA stretching in time the these improvements Despite Ancient DNA (aDNA) is a low quality and low quantity source of genetic material, material, genetic of source quantity and low a quality low (aDNA) is DNAAncient Since Method D was developed, several extraction protocol modifications have been have been modifications protocol extraction several D was developed, Method Since Author and petrous tooth on protocols extraction DNA of two performance the weHere test Manuscript This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology John Wiley&Sons, Inc. 2
pre digestion (between 15 and 30 minutes) with an EDTA and proteinase K buffer wasK buffer EDTA and proteinase an with minutes) and 30 15 (between pre digestion temperate or cold contexts (Barlow et al., 2016; Boessenkool et al., 2016; Dabney et al., 2013a; al., 2013a; Dabney et et al., 2016; Boessenkool (Barlow et al., 2016; contexts cold temperate or majority of development in aDNA extraction protocols has been conducted with samples from samples with conducted has been protocols aDNAin extraction majority of development decalcification step, results in improved endogenous aDNA recovery. In addition, because In the addition, aDNA recovery. endogenous improved in results step, decalcification (henceforth Method H), with an initial EDTA wash and an extended digestion and digestion and an extended EDTA wash an initial with H), Method (henceforth and characterize differences in base pair composition, base pair in differences and characterize microparticles from dental calculus than hydrochloric acid. acid. hydrochloric calculus than dental from microparticles endogenous reads, recovered after shotgun Illumina sequencing from parallel, paired extractions paired extractions from parallel, Illumina sequencing shotgun after recovered reads, endogenous included. included. Tromp et al. (2017) who observed that EDTA decalcification was more effective at recovering at recovering effective was more decalcification observed EDTA that et al. (2017) who Tromp and petrous portion samples recovered from tropical sites. Here we examine raw DNA yields and DNA rawwe Here examine sites. from tropical recovered samples portion and petrous Mexico (n=6). Additionally, one historic sample from a Tanzanian chimpanzee was also was also chimpanzee a Tanzanian from sample (n=6). one historic Additionally, Mexico on mineralized dental calculus without significant DNA loss. This finding was mirrored by finding This DNA loss. significant without calculus dental mineralized on Pinhasi et al., 2015; Tromp et al., 2017), this work focuses on protocol optimization with with tooth work optimization focuses protocol on this et al., 2017), Tromp et al., 2015; Pinhasi Age sites from Puerto Rico (n=5) and one tomb from the Maya site of Yaxuna in Yucatán, in of Yaxuna Mayathe site from and one tomb (n=5) Rico from Puerto Age sites Warinner et al. (2014) used an initial EDTA wash to remove loosely bound surface contaminants surface contaminants bound removeto loosely EDTA wash an used initial et al. (2014) Warinner Gamba et al., 2016; Gamba et al., 2014; Glocke & Meyer, 2017) but see (Damgaard et al., 2015; et al., 2015; (Damgaard Meyer, see & but 2017) Glocke et al., 2014; Gamba Gamba et al., 2016; samples included in this research were obtained from human remains excavated at three Ceramic at three excavated remains human from research obtained were this in included samples extractions of petrous portion tissue (Gamba et al., 2014; Pinhasi et al., 2015). Likewise, et al., 2015). Pinhasi 2014; (Gamba et al., tissue portion of petrous extractions damage profiles, and average read lengths recovered between the two methods. Archaeological Archaeological methods. two the between recovered lengths read and average damage profiles, Tissue samples from petrous portions and/or teeth were obtained from six human skeletons skeletons human from six were teeth obtained and/or portions from petrous samples Tissue extraction protocols designed by Richards et al. (1995) and was also recently reported in in reported recently also and was (1995) et al. designed Richards protocols by extraction inconclusive results in statistical tests conducted with this tissue. tissue. this with conducted tests statistical in results inconclusive endogenous aDNA. The use of similar detergent solutions has been implemented previously in in previously has been implemented solutions detergent use of similar The aDNA. endogenous applicable to tooth samples, since the small sample of petrous portions obtained led to to led obtained portions of petrous sample since small the samples, tooth applicable to
most of the archaeological samples had extremely low had extremely samples of archaeological the most Gamba et al. (2016)
successful in reducing proportions of exogenous, contaminant DNA and in enriching extracts for extracts enriching DNA and in contaminant of exogenous, proportions reducing successful in clonality, while libraries sequenced from Method D e from Method sequenced libraries clonality, while tropical environments. However, one important caveat of our study is that these findings are only are these findings that is caveat of our study important one However, environments. tropical and resulted in increased aDNA yields. Similarly, Damgaard et al. (2015) observed that a brief a brief that et al. (2015) observed Damgaardyields. Similarly, aDNA increased and in resulted H for maximized recovery of informative ancient DNA molecules from remains buried in in buried remains DNA from molecules ancient recoveryof informative H for maximized EDTA, proteinase K and N laurylsarcosyl detergent solution, aided in solubilizing cell proteins cell proteins solubilizing aided in solution, detergent N laurylsarcosyl K and EDTA, proteinase DNA fragment sizes were under 80 bp, we conclude that Method D is better suited than Method Method than suited better D is Method that conclude we under bp, were 80 sizes DNA fragment Libraries sequenced from Method H extracts had highe H extracts Method from Libraries sequenced rubble and fill falling from the ceiling of the chamber. In most skeletons, only one tissue type, type, one tissue only In skeletons, chamber. most ceiling the of the from falling rubble and fill the 6 the an originally unfilled burial space that dates to the Maya Early Classic period (specifically from (specifically the period Maya to Early Classic space dates that burial an originally unfilled excavated from a single tomb in the archaeological site of Yaxuna in Yucatán, Mexico. This was This Mexico. Yucatán, in of Yaxuna site archaeological the in a single tomb from excavated th century A.D.) During the centuries of deposition, the burial space gradually filled with with filled gradually space burial the of deposition, century centuries the During A.D.) In this study, we evaluate whether using a modified version of the Method D protocol D protocol of Method the version a whether modified using evaluate weIn study, this Study results suggest that both methods were similarly efficient at aDNA recovery. recovery. at aDNA efficient were methods similarly both that suggest results Study Samples were collected from skeletal remains excavated from three tropical contexts. contexts. three tropical excavated from remains skeletal collected were from Samples
Author Manuscript found that a secondary digestion and decalcification digestion a secondary that found This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology M ATERIALS AND METHODS AND METHODS ATERIALS Sample and site information information site and Sample John Wiley&Sons, Inc. 3 xtracts have shorter DNA fragments. Since Since fragments. DNA have shorter xtracts sequence read complexity, read sequence average and average (<5%), content endogenous r endogenous content but also higher also but content endogenous r using lysis buffer with with buffer lysis using post mortem post mortem Page 4of24
Page 5of24
published guidelines (Kircher et al., 2012; Seguin Orlando et al., 2015). Indexed libraries were libraries were Indexed et al., 2015). Seguin Orlando al., 2012; et guidelines (Kircher published potential contamination. contamination. potential petrous portion or teeth was available for sampling, while only two individuals (AD 372 and (AD 372 individuals two only while for sampling, was available or teeth portion petrous schweinfurthii protocols including an initial EDTA wash as in Warinner et al. (2014), an extended digestion and digestion an extended et al. (2014), EDTA Warinner wash as in including an initial protocols was determined via qPCR using the KAPA Library Quantification kit following manufacturer following manufacturer kit LibraryQuantification using KAPA the qPCR was via determined purified using the Qiagen MinElute PCR purification kit, and DNA content after amplification amplification after purification and DNA content kit, PCR MinElute Qiagen usingpurified the Method H. Method D was implemented as in (Dabney et al., 2013a) with the modification that that modification the with et al., 2013a) (Dabney as in implemented D was H. Method Method indexing cycles. All libraries were double indexed and amplified for 11 25 cycles following cycles following for 11 25 and amplified indexed double were libraries cycles. All indexing (qPCR) with the 2X Dynamo SYBR Green qPCR Master Mix to determine the ideal amount of amount ideal determine the to Master Mix qPCR Green SYBR Dynamo 2X the with (qPCR) Ceramic Age contexts, between A.D. 500 1300 (Pestle, 2010; Pestle & 2012). Pestle Colvard, 2010; (Pestle, 500 1300 between A.D. contexts, Ceramic Age and including negative controls. DNA content in the libraries was quantified using real time PCR PCR using real time quantified the libraries was in DNA content controls. and including negative and between uses (Cooper & Poinar, 2000; Gilbert et al., 2006). et al., 2006). Gilbert & 2000; Poinar, (Cooper uses and between Paso del Indio (n=2), and Punta Candelero (n=2). All five individuals date from pre contact five individuals All Candelero (n=2). Punta Indioand (n=2), del Paso ten teeth and four petrous portions. portions. petrous and teeth ten four here] 1 [Table GB 7. After death, MacDee’s remains were stored in a in stored were remains MacDee’s GB 7. death, After full body coverings, bleach decontamination and UV irradiation of tools and work area before and UV of tools irradiation and decontamination bleach bodyfull coverings, collected from humans remains excavated from three open air sites in Puerto Rico: Tibes (n=1), Tibes Rico: Puerto in sites open air from three excavated remains humans collected from the Puerto Rican or Tanzanian remains. In total, 12 individual skeletons were producing skeletons sampled individual In 12 remains. total, or Tanzanian Rican Puerto the
western Tanzania (prior to Gombe becoming a national becoming Gombe (prior to western Tanzania insects and the skeleton was subsequently cleaned. cleaned. was subsequently and skeleton the insects one tooth was collected from the skeletal remains of remains skeletal the from was collected one tooth
(2015). All laboratory procedures were conducted using contamination controls, such as such use of controls, using contamination conducted were laboratory procedures (2015). All AD 373) could be sampled in both anatomic locations (Table 1). Additionally, five teeth were were five teeth (Table 1). Additionally, locations anatomic both in sampled AD 373) be could Schuenemann et al. (2011). Petrous portions were sampled as recommended by Pinhasi et al. by Pinhasi as recommended sampled were portions Petrous al. (2011). et Schuenemann
Extraction blanks were included throughout the proce throughout the included were blanks Extraction et al., 2013). (Simbolo S1) The roots were covered in aluminum foil and pulverized by blunt force with a hammer as in with force by and pulverized blunt foil aluminum in covered were The roots DNA yields through fluorometric quantification with the Qubit 2.0 High Sensitivity assay (Table assay High 2.0 Sensitivity Qubit the with quantification yields fluorometric through DNA Crosslinker. Teeth were sliced transversally at the cemento enamel junction using a Dremel tool. tool. using a Dremel junction cemento enamel at the transversally Teeth sliced were Crosslinker. of bone or tooth powder were used for each extraction. 1 µl of each extract was used to measure measure was to used of each µl 1 extract extraction. each forwere used powder of or bone tooth and samples were UV irradiated for 5 minutes on each side in a UVP CL 1000 Ultraviolet Ultraviolet CL 1000 UVP each a in side on for minutes 5 UV were irradiated and samples the TET buffer was warmed to 65 65 to warmed was TETbuffer the al., 2013a). See Supplementary File S1 for complete Method H protocol. Approximately 100 mg mg 100 Approximately H protocol. Method complete for File S1 al., 2013a). See Supplementary The outer surface was mechanically removed with a Dremel tool (Rohland & Hofreiter, 2007) & 2007) Hofreiter, a (Rohland Dremel tool removed with was mechanically The outer surface decalcification step as in Gamba et al. (2016), and binding and purification steps as in (Dabney et (Dabney as in steps andbinding purification and Gamba et al. (2016), as in step decalcification and inhibitors, tooth and bone samples were cleaned with a 1% sodium hypochlorite solution. solution. hypochlorite a 1% sodium with cleaned were and samples bone tooth and inhibitors, Ancient DNA Laboratory, a Class 10,000 clean room facility. To eliminate surface contaminants surface contaminants eliminate facility. To clean room 10,000 a Class Laboratory, DNAAncient Each sample was extracted twice, one time using Method D and a second time using time D and aMethod second using twice, one time extracted Each was sample Sample processing and DNA extractions were conducted at the Arizona State University State Arizona at the conducted were extractions and DNA processing Sample Double stranded libraries were built following the protocol by Meyer and Kircher (2010) (2010) and Kircher by protocol Meyer the following were built stranded libraries Double
Author Manuscript) who died in 1966. The chimpanzee was named McDee by scientific observers in in observers scientific by was The named McDee chimpanzee 1966. in died who This article isprotected by copyright. All rights reserved. Sample processing and DNA extraction and extraction DNA processing Sample American JournalofPhysicalAnthropology Library preparation and sequencing and sequencing preparation Library ° C in a heat block. Method H combines steps from earlier steps H combines a Method heat in block. C
John Wiley&Sons, Inc. 4 Petrous portion tissue was not available from available was not tissue portion Petrous a wild chimpanzee ( chimpanzee a wild metal box. Flesh was eaten away Flesh was eaten by box. metal park) and is referred to in this study this in as to park) referred and is Pan troglodytes troglodytes Pan ss to monitor monitor to ss Lastly,
paired sample treatment combinations extrapolation e extrapolation combinations paired sample treatment produced on a single flow cell of the Illumina HiSeq flow cell of the single produced a on v2.0 (Daley & Smith, 2013) on downsampled BAM files containing all mapped reads. reads. containing mapped all BAM files downsampled on 2013) & Smith, (Daley v2.0 probability of a DNA fragment terminating in a single stranded overhang ( overhang a single stranded in fragment terminating a DNA ofprobability preseq patterns were characterized using mapDamage v.2.0.2 (Ginolhac et al., 2011; Jónsson et al., Jónsson (Ginolhac et al., 2011; v.2.0.2 using mapDamage characterized were patterns and rescaled damage were BAM Li files et al., 2009). (H. 0.1.19 v. SAMtools with performed sequence read processing. processing. read sequence (Okonechnikov et al., 2016). See Supplementary File S2 for additional details of shotgun details forFile additional S2 Seeet Supplementary al., 2016). (Okonechnikov complexity ( the DNA 1000 assay on the Agilent 2100 Bioanalyzer. Heteroduplexes that arose during that Bioanalyzer. Heteroduplexes 2100 Agilent the assay DNA on the 1000 the mitochondria replaced by the revised Cambridge Reference Sequence (rCRS) (Andrews et (rCRS) Sequence Reference Cambridge by revised the replaced mitochondria the (https://github.com/jstjohn/SeqPrep). (https://github.com/jstjohn/SeqPrep). percent endogenous content, library content, endogenous percent coordinates), external has unique that read sequence treatments and to control for differences in sequenc in for differences control and to treatments Extraction yields (ng/µL), number of mapped, unique reads of unique number mapped, yields (ng/µL), Extraction Extrapolation curves were calculated in in curves calculated were Extrapolation instructions (Kapa Biosystems). Fragment analysis of the indexed libraries was conducted with with was conducted libraries of indexed the analysis Fragment Biosystems). (Kapa instructions (hg19)reference with GRCh37 the mapped to were reads downsampled the samples, human the MarkDuplicates module v.2.12.1 within Picard Tools ( Tools Picard within v.2.12.1 MarkDuplicates module observing cytosine deamination in a double strand ( strand a double in deamination cytosine observing trimming and merging: GB 7: 5,192,848 reads and PI 6 and reads GB 7: 5,192,848 and merging: trimming matching the lowest number of reads obtained per sam per obtained of number reads matching lowest the quality filtering were repeated for these data using these data for repeated were quality filtering pa default with seqtk using samples all selected for and extrapolating to 600,000,000 total reads. This i reads. This total 600,000,000 to and extrapolating S number of reads: GB 7 and PI 67. For these two sampl two For PI 67. these and GB 7 ofnumber reads: (measured as fraction of the mapped reads that are d are that reads mapped of the as fraction (measured Quality filtering ( Quality filtering and conditions. primers PCR for Genomic Analysis. See Supplementary File S1 for additional details of library preparation, library preparation, of details forFile additional S1 See Supplementary for Analysis. Genomic misincorporations at first position, probability of G to A misincorporations at last position, position, at last probability A misincorporations G to of position, at first misincorporations 2013). Parameters examined included deamination patterns, probability of C to T to C patterns, of probability deamination included examined Parameters 2013). deamination in a single strand context ( context a single in strand deamination two runs on the Illumina HiSeq 2500 2500 Illumina HiSeq the on runs two recommendations by Schubert et al. (2014). Schubert by recommendations quantified as detailed above. Reconditioned shotgun libraries were sequenced on one lane across one lane on sequenced libraries were shotgun Reconditioned above. as detailed quantified Mapping was performed using BWA v. 0.7.5 (Heng Li & Durbin, 2009) following following & Li 2009) Durbin, (Heng using 0.7.5 v. BWA performed Mapping was indexing were eliminated through reconditioning PCR and all libraries were purified and re and purified libraries were and all PCR through reconditioning eliminated wereindexing al., 1999). For the chimpanzee samples, the reads were mapped to the PanTro4 assembly. assembly. PanTro4 the were to mapped reads the samples, chimpanzee Foral., 1999). the ummary statistics were estimated on rescaled BAM files using Qualimap v.2.2.1 v.2.2.1 files using Qualimap BAM rescaled on estimated were ummary statistics
Future experiment yield predictions were calculated calculated yield were predictions Future experiment
Illumina sequence reads were merged and adapters trimmed using SeqPrep using SeqPrep trimmed and adapters were reads merged Illumina sequence
percent distinct reads as measured by the the by as measured reads distinct percent Author Manuscript ≥ Q30), removal of duplicates and of reads with multiple mappings multiple was with of reads and removal of duplicates Q30), This article isprotected by copyright. All rights reserved. Shotgun read mapping and processing and processing read mapping Shotgun American JournalofPhysicalAnthropology (2 x 100 bp reads) at the Yale Center Yale at the reads) Center bp 100 Run (2 x mode Rapid in To compare sequencing results directly extrac across results sequencing compare To Statistical Analyses Analyses Statistical John Wiley&Sons, Inc. δ p S reseq ). Library complexity estimates were generated usinggenerated were estimates Librarycomplexity ). Duplicate reads were identified using the identified were Duplicate reads using the default step size parameter ( s 1000000) ( s parameter 1000000) size step default the using 5 the same parameters listed above. listed same the parameters (https://github.com/lh3/seqtk). For (https://github.com/lh3/seqtk). rameters s the maximum number of paired end reads reads of number paired end maximum the s er output, 1 million reads were randomly randomly were reads million 1 er output, δ uplicates: number of duplicate reads / / reads of number duplicate uplicates: 2500 in Rapid Run other all Run Formode. Rapid in 2500 D stimates failed due to low read low depth. failed due to stimates ), and probability of observing cytosine cytosine observing and of probability ), p ple treatment combination after adapter after combination ple treatment http://broadinstitute.github.io/picard es, random downsampling was repeated was repeated es, downsampling random reseq 7: 6,876,556 reads. Read mapping and Read mapping reads. 6,876,556 7: for the two libraries with highest with for libraries two the ), clonality function ccurve (defined as a mapped as a mapped (defined λ ), probability of tion tion ).
Page 6of24
Page 7of24
parameters were compared for each sample across extraction treatments by using paired T tests Ttests paired by treatments using extraction across for sample each compared were parameters base graphics (R Core Team, 2016). 2016). Core Team, graphics (R base plots and figures were generated using the ggplot2 (H Wickham, 2009), gridExtra (Auguie,gridExtra 2009), (H Wickham, ggplot2 generated the and using figures were plots but not petrous portion samples (Table 2). The relat (Table samples 2). The portion petrous not but between 0% to 14%, and tended to be higher for Metho for be higher tended to and between 14%, 0% to this project are available at: at: available project arethis Computing Saguaro environment. All calculations were performed in R 3.2.4. Scripts written for written Scripts 3.2.4. R in were performed calculations All Saguaro environment. Computing cor.test function. R the in implemented
(2012) (Figure S1 and S2). Correlations between variables were tested using Pearson’s r using Pearson’s tested were variables between Correlations and S2). S1 (2012) (Figure histograms of the difference between paired values as recommended by Ghasemi and Zahediasl Zahediasl and Ghasemi by as recommended values paired between histograms of difference the normality test (Table S3) and through visual examination of Quantile Quantile plots and plots of Quantile Quantile examination visual and through S3) (Table normality test sequencing. sequencing. according to type of tissue. Normality assumptions were evaluated using a Shapiro Wilk Shapiro Wilk a evaluatedwere using assumptions Normality tissue. of according type to or non parametric Wilcoxon signed rank tests. For these analyses, samples were subdivided were subdivided samples signed these analyses, For rank tests. Wilcoxon or non parametric filtering) over the total amount of down sampled reads (Table S2). Most samples had <5% samples Most reads S2). sampled of down (Table amount filtering) total the over here] 2 [Table difference may be attributable to the younger age of the historic chimpanzee sample. sample. chimpanzee of historic younger the age the to attributable difference be may as the proportion of unique reads mapping to the reference (after duplicate removal and quality removal and quality (after duplicate reference the to reads mapping of unique as proportion the samples extracted with either method irrespective of tissue (Table 2). (Tableof 2). tissue irrespective method either with extracted samples content, an up to sixteen fold higher content than that found in the human libraries. This libraries. This human the in found than that content higher sixteen fold an to up content, (Figure S3). These analyses did not reveal significant differences in mean DNA yields between DNA mean differences in reveal not significant did analyses (Figure These S3). samples extracted with Method H tended to have highe have H tended to Method with extracted samples
and here] 2 [Figures 1 DNA yields were evaluated through flourometric quantifications of raw extracts (ng/µL) (ng/µL) extracts of raw quantifications flourometric through yields evaluated were DNA R or with packages 2007) Wickham, (Hadley 2 and reshape 2016) (H Wickham, tidyr2016), reads mapping to the reference post quality filterin reference the post quality to reads mapping were statistically significant for teeth but not pet not for but teeth significant were statistically number of mapped reads), reads), of number mapped
Percent clonality was found to be diff significantly to found was Percent clonality
million randomly selected reads for all samples all reads randomly for selected million e
ndogenous content, except for the chimpanzee sample, GB 7, which yielded >10% endogenous endogenous yielded >10% GB 7, which sample, for chimpanzee the except content, ndogenous This research was conducted using resources from the ASU High Performance High the ASU Performance from resources using conducted research wasThis For each DNA library, between 1 and 27 million reads were obtained after shotgun after reads obtained were million and 27 1 library, between For DNA each Sequence clonality (measured as fraction of mapp the as fraction clonality (measured Sequence
Authorwere performed analyses For statistical consistency, Manuscript This article isprotected by copyright. All rights reserved. Endogenous content and library complexity complexity and content library Endogenous percent GC content, average fragment lengths, and da and lengths, fragment average content, GC percent Computational resources and R packages R resources and Computational https://github.com/mnievesc/aDNAExtMethodsPaper_scri American JournalofPhysicalAnthropology John Wiley&Sons, Inc. DNA yields yields DNA R ESULTS ESULTS . Percentage endogenous content was calculated was calculated content endogenous . Percentage 6 rous portion samples (Table 1). 1). (Table samples portion rous erent between extraction methods for teeth, methods extraction erent between g (Figure 1, Figure S4). These differences These differences S4). Figure g 1, (Figure ionship between clonality and endogenous endogenous between clonalityand ionship r endogenous content and more unique and more unique content endogenous r d H libraries (Figure 2, Figure S5). S5). Figure 2, H libraries (Figure d ed reads that are duplicates) ranged ranged duplicates) are that ed reads with a starting number of one number a starting with
mage Overall, Overall, pts as . All . All
predicted to yield a higher amount of complex DNA fragments with deeper sequencing (up to (upsequencing to deeper DNA fragments with yield of complex amount a higher predicted to bp. This small size is consistent with expectations for degraded remains (Briggs et al., 2007; et al., 2007; (Briggs expectations for remains consistent degraded with is size small This bp. (Figure 4A). Overlaid plots showing the length distr showing length the plots 4A). (Figure bp) Overlaid 72.07 portion: proportion of larger fragments (Figure S8 10). Although this pattern of higher average fragment fragment average pattern of higher this Although (Figure S8 10). fragments of larger proportion a higher H libraries had Method that filtering, demonstrate and mapping and after beforeboth proportion of distinct reads that map to the same sites and therefore may have a strong bias and bias a strong and have therefore same may the sites to map that reads of distinct proportion (Daley & Smith, 2013). Therefore, 2013). &(Daley Smith, amount of sequence data generated, and can give false estimates with low amounts of reads amounts low with give estimates false and can generated, data of sequence amount sequencing effort with the same libraries. This extrapolation analysis is highly sensitive to the the to highly sensitive is analysis extrapolation This same libraries. the with effort sequencing research with tropical samples (Kehlmaier et al., 2017; Schroeder et al., 2015). Samples Samples et al., 2015). Schroeder2017; et al., (Kehlmaier samples tropical research with libraries, we used the c_curve the libraries, we used Dabney et al., 2013b; Meyer et al., 2014) and similar to sizes obtained in previous aDNA previous in obtained sizes to and similar Meyer et al., 2014) al., 2013b; Dabney et either tissue type type either tissue Figure 3 demonstrates that, in both cases, libraries constructed with Method H extracts were H extracts Method constructed with cases, libraries both in that, Figure demonstrates 3 sample. No significant correlation was found between endogenous content and read length in in and read length content endogenous between found was correlation significant No sample. than those extracted with Method H Method with extracted those than bp) 53.65 portion: Petrous S7 [Figure here] 3 libraries created from Method D extracts. D extracts. from Method libraries created
finding is influenced by low statistical power due to the smaller size of the petrous portion portion petrous of the size smaller the power due to statistical low by influenced finding is H libraries, irrespective of tissue type, but this difference was not statistically significant ( significant statistically was not difference type, this tissue but of H libraries, irrespective = 0.1265, t = 0.5409, df = 18, p=0.5952; Petrous portions: Pearson’s r portions: Petrous = p=0.5952; 18, = df = 0.1265, t 0.5409, r (Teeth: Pearson’s tissue for either It significant linear or was not S6A. Figure in shown is content (Figure df S11A). = p=0.5263) 6, 0.6724 = t = 0.2647, Pearson’s r portions: Petrous df = p=0.5881; 18, 0.5514, = t = 0.1288, 1.003, df = p=0.3546 1.003, 6, df = 6, p=0.5559). df = p=0.5559). 6, Pearson’s this difference was only found to be statistically significant in teeth (Figure 4A). We suspect this this suspect 4A). We (Figure teeth in significant be statistically to difference found was only this method. Method D libraries had a slightly higher mean proportion of distinct reads than Method reads Method than of distinct mean proportion higher had a slightly D libraries Method method. Pearson’s combinations in the two libraries that yielded the h libraries that two the in combinations lengths in Method H libraries is evident in boxplots for both tooth and petrous portion samples, samples, portion and petrous tooth for both boxplots in evident is H libraries Method lengths in high redundancy (Head et al., 2014). In this dataset, complexity was high regardless of extraction of extraction regardless complexity was high In dataset, this al., 2014). et (Head high redundancy 600 million reads). In both cases, saturation of the In reads). cases, saturation both million 600 shown in in shown covered with a single sequencing experiment. In contrast, low complexity libraries have a large a large have In complexity libraries low contrast, experiment. sequencing a single with covered [Figure 4 here] [Figure here] 4 that map to different parts of the reference genome. Therefore, more parts of the reference are of reference the more parts genome. Therefore, of reference the different to map parts that recovered for each library. High complexity libraries have a large proportion of distinct reads of distinct have proportion a large libraries complexity High library. each recovered for extracted with Method D yielded smaller average DNA fragment sizes ( sizes DNA fragment yielded average smaller D Method with extracted
). The relationship between complexity and endogenou and complexity between ). The relationship All samples, irrespective of extraction method had average DNA fragment lengths <100 lengths fragment average DNA had method of extraction irrespective samples, All T W o examine this question further and test which method produced higher complexity complexity produced higher method which question further and this test examine o Figure S6B e lc_extrap the used r r
Author Manuscript (Teeth: Pearson’s r (Teeth: . No significant correlation was observed between th observed between was correlation significant No . ) .
This article isprotected by copyright. All rights reserved. DNA fragment lengths and GC content content and GC lengths fragment DNA function in preseq to estimate the number of distinct reads distinct of number the estimate to preseq in function function within preseq to predict the expected yieldfor a larger preseq expected predict the to within function American JournalofPhysicalAnthropology this analysis was only possible for sample treatment for was only possible analysis this
= 0.3321 t = 1.4936, df = 18, p=0.1526; Petrous portions: portions: Petrous df = p=0.1526; 18, 1.4936, = t = 0.3321 John Wiley&Sons, Inc. 7 complexity curve is reached earlier for earlier reached is complexity curve PI 67 and GB 7. and GB 7. PI 67 reads: ighest of number s content in the tested libraries is libraries is tested the in content s (Tooth: 77.22 and Petrous and Petrous 77.22 (Tooth:
ibution of sequence reads, reads, of sequence ibution Tooth: 58.63 bp and bp 58.63 Tooth: = 0.2466, t = t 0.6234, = 0.2466, (Teeth: (Teeth: values two e = 0.3789, t = t = 0.3789,
Figure Page 8of24
Page 9of24
preserved tooth and petrous portion remains excavated from tropical contexts. Our experimental Our experimental contexts. from tropical excavated remains portion and petrous tooth preserved between samples extracted with either extraction method either extraction with extracted between samples probabilities (>0.70) probabilities portion sample likely resulted in low statistical power to detect significant differences. Because differences. power significant statistical low detect in to likely sample resulted portion (Boessenkool et al., 2016; Damgaard et al., 2015) Damgaard et al., 2016; (Boessenkool Dabney et al., 2013b; Overballe Petersen et al., 2012). et al., 2012). Overballe Petersen al., 2013b; Dabney et digestion or pre digestion wash steps also had negligible effects on DNA damage profiles damage profiles DNA on effects had negligible also wash steps ordigestion pre digestion consistent with known damage patterns of authentic aDNA sequences (Briggs et al., 2007; et al., 2007; (Briggs aDNA sequences authentic damage of known patterns with consistent exhibit different postmortem damage patterns. Other studies have demonstrated that modifying modifying that have demonstrated studies Other damage patterns. different postmortem exhibit and last position of each fragment (see Supplementary File 3 for damage plots). This is is This plots). damage File for 3 Supplementary fragment of each (see position and last who found that ancient samples extracted with several silica based extraction methods did not not did methods extraction silica based several with extracted ancient samples that found who fragments. This finding is consistent with results previously reported by Gamba et al. (2016), et al. (2016), Gamba reported by previously results with consistent is finding fragments. This results suggest that both Method D and Method H successfully recovered degraded genetic degraded recovered successfully H D and Method Method suggest both results that content ( content
The latter suggests that neither method is biased ag biased is neither method that suggests reads. The latter hi or reads of sequence populations assuch multiple a of the Coverage A misincorporations). Tor G to to observed in raw DNA yields, library complexity or po yields, library complexity DNA raw observed in However visual examination of BAM files did not reve not of BAM did files examination visual However = 0.6578, t = 2.1395, df = 6, p=0.0762) (Figure S11B). (Figure df = p=0.0762) 6, = = t 2.1395, 0.6578,
not found to be statistically significant significant be statistically to found not read depth for confident assessment (Fu et al., 2013 (Fuassessment for confident read depth insufficient for contamination determination with Ba with determination for contamination insufficient Neither of the three DNA damage patterns examined ( examined damage patterns DNA Neither of three the
endogenous content, clonality and number of mapped, of mapped, and number clonality content, endogenous r found between average DNA fragment length and GC content for all samples (Teeth: for content samples all GC and length DNA fragment average between found (petrous portion) and AD 377 H (tooth) had the lowes had the (tooth) AD 377 H and (petrous portion) = 0.5585, t = 2.8566, df = p=0.01048 18, = df = t 2.8566, 0.5585, generated with each method for both tissues (Figure 4C). A significant negative correlation was correlation negative A significant (Figure 4C). tissues for both method each with generated
df = p=0.0448 6, fragment lengths versus average percent GC content clearly distinguishes between samples clearly distinguishes content GC average percent versus fragment lengths types but were only significant for comparisons with tooth extracts. The small size of the petrous of petrous the size extracts. The small tooth with comparisons for significant types only were but with Method D, irrespective of tissue. These differences were visible in boxplots for all tissue for tissue all boxplots in visible were These differences of tissue. D, irrespective Method with fragments recovered with Method H were, on average, 19 base pairs longer than those recovered those than longer base 19 pairs average, on H were, Method with fragments recovered material from tooth and petrous portion samples. samples. portion and petrous from tooth material datasets and note that further research with more comprehensive samples of petrous portion portion petrous of samples more comprehensive with research further that anddatasets note of this, we refrain from extrapolating meaningful conclusions based on the petrous portion portion petrous based the on conclusions meaningful extrapolating we from refrain of this,
In this research, we explored the performance of two extraction protocols on poorly on protocols extraction of two performance the explored we In research, this this difference was difference this but of tissue, irrespective content GC D libraries had higher Method
Significant differences between methods were observe were methods between differences Significant Author Manuscript r Pearson’s Teeth: ). No significant correlation was observed between p between was observed correlation significant No ). of C to T and G to A misincorporations caused by DNA by caused A misincorporations Tand G to to of C This article isprotected by copyright. All rights reserved.
= 0.4092, t = 1.903 df = p=0.0732 18, = df = t 1.903 0.4092, American JournalofPhysicalAnthropology John Wiley&Sons, Inc. (Table 2, Figure 4B). A scatterplot of average DNA of average DNA Figure A scatterplot 4B). 2, (Table DISCUSSION DISCUSSION DNA damage damage DNA ; Petrous portions: Pearson’s portions: Petrous ; 8 No statistically significant difference was difference was significant statistically No .
; Racimo et al., 2016; Renaud et al., 2015). et al., 2015). Renaud et al., 2016; Racimo ; gh mismatch rates. rates. gh mismatch utosomal and mitochondrial genomes were were genomes and mitochondrial utosomal yesian tools which require >3 5X minimum >3 5Xyesian require minimum which tools stmortem damage patterns in shotgun in damage patterns stmortem t damage patterns (~0.50 probability of C of probability C (~0.50 damaget patterns unique reads (post quality filtering) (post quality reads unique Replicates PC 117 D (tooth), AD 373 H AD 373 H (tooth), PC 117 D Replicates al patterns consistent with contamination, contamination, with consistent al patterns ( ainst recovery of degraded DNA degraded of recovery ainst
Figure S12 = 0.7181, t = t 2.5278 0.7181, = λ in DNA fragment length, fragment DNA in d ,δ D, δ ; Petrous portions: Pearson’s portions: Petrous ; S ) differed significantly significantly ) differed Most samples had high samples ). Most ercent GC and endogenous and endogenous GC ercent r
damage at the first first at the damage Pearson’s Pearson’s . DNA . DNA r
bone extractions performed with short digestions. digestions. short with performed extractions bone predicted library complexity with the two best preserved samples (PI 67 and GB 7) indicated that that GB 7) indicated and (PI 67 preserved samples best two the with complexity predicted library (2016) found that mean aDNA fragment lengths were smaller and GC content was higher in was in higher content and GC smaller were lengths fragment aDNA mean that (2016) found aDNA recovery with digestion steps longer than one hour. More recently, Boessenkool et al. Boessenkool recently, Morehour. one longer steps than digestion with aDNA recovery characteristics of endogenous aDNA. For instance, Damgaard et al. (2015) observed diminished observed diminished et al. (2015) Damgaard For aDNA. instance, endogenous of characteristics methods have suggested that digestion time may be a strong influence on recovery rates and rates recovery on a influence strong be may time digestion that have suggested methods EDTA. Other with calculus samples washes of yields after pre extraction DNA raw in reductions interferes with recovery of short DNA fragments. In general, reports comparing aDNA extraction extraction aDNA In comparing reports general, DNA fragments. of short recovery with interferes observe not did et al. (2014) step. Warinner and decalcification digestion extended during the identify where small DNA fragments are being lost in the extraction process. process. extraction the in lost being fragments are DNA identify small where Korlevic et al., 2015). But a more recent report by Glocke and Meyer (2017) found that EDTA that found (2017) Glocke and Meyer by a report more recent But Korlevic et al., 2015). EDTA wash or pre digestion may the occurred during loss have DNA fragment D, short Method separate library preparation and sequencing of EDTA wash and pre digestion fractions to fractions to wash and pre digestion EDTA and of sequencing library preparation separate steps or bleach based decontamination washes (Damgaard et al., 2015; Gamba et al., 2016; et al., 2016; Gamba washes (Damgaard et al., 2015; or decontamination bleach based steps in implemented steps and purification same the binding H uses Method al., 2013a). that Given then further kept overnight at 37 overnight furtherthen kept needed to avoid loss of small DNA fragments. Future optimization efforts may also benefit from efforts benefit from also may optimization Future DNA fragments. of small loss needed avoid to Examination of the complexity curves for both method for both curves of complexity the Examination 2013). & Smith, DNA fragment lengths recovered after modifying extraction procedures with extended digestion digestion extended with procedures extraction modifying after recovered lengths DNA fragment two part digestion step in which bone powder is first incubated for one hour in lysis buffer, and lysis buffer, in hour incubated for one first is powder bone which in step two part digestion solution and of subsequent digestion conditions, such as temperature and incubation time, are time, and incubation such as temperature conditions, digestion and of subsequent solution examined (Table S2). examined significant trends observed in tooth samples only. only. samples tooth in observed significant trends Low amounts of reads can lead to false estimates due to uncertainty of the extrapolation (Daley extrapolation of uncertainty due the to estimates false lead to can of reads amounts Low samples also had higher clonality. In other words, M In words, clonality. other had higher also samples s and sequence reads with the same starting and ending samethe starting with reads and sequence unique reads after quality filtering that were recovered with Method H in the two samples samples two the H in Method with recovered filtering were that after reads quality unique tissue is needed to resolve this question. The following discussion focuses on statistically statistically on focuses The following discussion question. this needed resolve is to tissue
content and more reads mapping to the reference afte reference the mapping to reads and more content despite the high clonality of Method H libraries, of Method clonality high the despite experiments. Complexity analyses are highly sensitive to the amount of sequence data generated. data of sequence amount the to highly analyses sensitive are Complexity experiments. with Method H versus Method D extracts. This pattern may be due to the higher number of of number higher the due may to pattern be This D extracts. H versus Method Method with Method H libraries would yield more unique DNA fragments upon repeated sequencing sequencing repeated fragments upon yield DNA more unique H libraries would Method percentage points higher in paired tooth samples ext samples tooth higher paired in points three percentage H. But all samples, irrespective of extraction method, showed a decrease in GC content with with content GC in a showed decrease method, of extraction irrespective H. samples, But all 2016). As GC content can be strongly affected by amplification enzymes used in the library the in enzymes used amplification affected by be strongly can content GC As 2016). correlated with lower contamination due to reduced presence of exogenous DNA (Racimo et al., et DNA (Racimo exogenous reduced presence of due to contamination lower with correlated 2017; Krause et al., 2010; Schuenemann et al., 2011). Higher GC content has also been has also content Higher GC et al., 2011). Schuenemann al., 2010; Krause2017; et ancient genomes composed of short DNA fragments (Briggs et al., 2007; Glocke & Meyer, Glocke (Briggs et al., 2007; fragments DNA of short composed genomes ancient that differential DNA preservation can cause compositional bias towards higher GC content in in content GC higher towards bias compositional cause can preservation differentialthat DNA larger average fragment size. This finding is consistent with previous research which has shown research previous has shown which with consistent finding is This size. average fragment larger tudies conducted with tooth and bone tissue also found no significant differences in average average in significant no differences found also and tissue bone tooth conducted with tudies We additionally found that libraries built from Meth libraries built that found additionally We E Method H implements an initial wash of 0.5M EDTA solution followed by an extended an extended by followed EDTA solution wash of 0.5M an initial H implements Method Lastly, although not a significant trend, we trend, we a significant not Lastly, although Author Manuscript (Dabney et fragments DNA ultrashort of Dfor was designed recovery Method xtraction
This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology ° C. It is possible that further optimization of the EDTA wash of the EDTA wash It further that optimization C. possible is John Wiley&Sons, Inc. 9 deeper sequencing would likely be most useful useful likely be most would sequencing deeper
observed that average GC content was at content average GC that observed ethod H libraries had more PCR duplicates duplicates more PCR H libraries had ethod r quality filtering. But, on average, on these But, r quality filtering. extrapolation of extrapolation However, coordinates. racted with Method D versus Method D versus Method racted Method with od H extracts had higher endogenous had endogenous higher H extracts od s demonstrates that that demonstrates s least least
Page 10of24
Page 11of24
protocols can take place, Method D continues to be the optimal choice for maximizing aDNA maximizing choice for be optimal the to D continues Method place, can take protocols preparation process (Aird et al., 2011; Dabney & Meyer, 2012; Seguin Orlando et al., 2015), all all et al., 2015), Meyer, Seguin Orlando & 2012; Dabney et al., 2011; (Aird process preparation cognizant of these recent advances, in this work we have focused on libraries built with the more the with built libraries on have work we focused this in advances, recent ofcognizant these increase endogenous aDNA yields (Barlow et al., 2016; Glocke & Meyer, 2017). While we are While 2017). Glocke & al., 2016; Meyer, yields (Barlow et aDNA endogenous increase towards ultrashort DNA fragment recovery with single stranded library preparation substantially substantially preparation library single stranded with fragment recovery DNA ultrashort towards recommendations by Wales et al. (2015) and focus on double stranded library protocols which which protocols on double stranded library focus et al. (2015) and Wales by recommendations Several recent studies have found that the combination of extraction methods geared methods of extraction combination the that found have Several studies recent samples. tropical preserved poorly assuch in complete mitochondrial DNA genomes (Nieves Colon et al., 2016). Thus, here we follow here follow et Thus, al., 2016). we genomes (Nieves Colon DNA mitochondrial complete DNA, may not necessarily lead to better results when read depth and coverage is inherently low, low, inherently is and coverage when read depth better results to lead necessarily DNA, not may samples from Puerto Rico contain sufficient endogenous DNA for effective enrichment of of enrichment effective DNA for endogenous sufficient contain Rico from Puerto samples alleviated somewhat by deep sequencing and high read depths, increased recovery of GC rich recovery of read increased depths, and high sequencing deep by somewhat alleviated al., 2013; Schroeder et al., 2015). Ongoing research in our laboratory has found that aDNA that found our laboratory has in research Ongoing 2015). et al., Schroeder al., 2013; suitable petrous portions and other tissues, such as dental calculus. A larger dataset shall further further dataset shall calculus. A larger dental as such and other tissues, portions petrous suitable alignment (Krause et al., 2010; Schuenemann et al., 2011). Although this problem may be may be problem Although this 2011). et al., Schuenemann 2010; et al., alignment (Krause for increasing informative sequence content with poorly preserved tropical samples (Carpenter et (Carpenter samples tropical poorly preserved with content sequence informative for increasing methodologies tailored for tropical aDNA samples will benefit from increased sampling of sampling benefit from increased will samples aDNA tropical for tailored methodologies content can also lead to low sequence coverage in aDNA due to difficulty with mapping and with aDNA in difficulty due to coverage sequence low leadalso to cancontent low levels of endogenous DNA (<2%). This suggests that enrichment approaches are essential essential are approaches enrichment that suggests DNA This (<2%). of endogenous levels low statements regarding each method’s performance with this tissue type. Future efforts to develop develop to type. efforts tissue Future this with performance method’s each regarding statements significant correlation between percent GC and endogenous content (Figure S11B). High GC GC S11B). High (Figure content endogenous and GC percent between correlation significant historic chimpanzee sample, all archaeological remains examined in this study very this in contained examined remains archaeological all sample, chimpanzee historic recovery in tropical tooth samples from ancient or historic contexts. contexts. historic or from ancient samples tooth tropical recovery in The smaller sample sizes obtained for petrous portion samples did not allow for conclusive for allow conclusive not did samples for portion obtained petrous sizes sample The smaller rich DNA, it may be better suited for ancient tropical samples. However, we did not identify a not did we However, samples. ancient tropical for suited be better may rich DNA, it commonly used double stranded library protocol (Meyer & Kircher, 2010). Except for the Except & 2010). Kircher, (Meyer library protocol stranded double used commonly Hofreiter et al., 2015). Therefore, our findings suggest that, until further optimization of new optimization further until that, findings suggest our Therefore, et al., 2015). Hofreiter glance, these results suggest that since Method D likely allowed for higher recovery rates of GC rates of recovery for higher allowed D likely since Method that suggest results glance, these Additionally, previous reports have demonstrated that extremely short molecules obtained after obtained molecules extremely short that have demonstrated reports previous Additionally, sequence content to come from small DNA fragments in ancient remains (Allentoft et al., 2012; et al., 2012; (Allentoft ancient in remains DNA fragments from small come to content sequence explain the observed differences in base composition between extraction treatments. At a At first treatments. between extraction composition base in observed the differences explain
are better suited for studies geared towards enrichment of multi copy organellar DNA. of multi copy organellar enrichment towards geared for studies suited are better PCR duplicates. In contrast, Method D recovered smal In D recovered Method duplicates. contrast, PCR exacerbated aDNA degradation that takes place in the tropics, we expect most informative informative most wethe tropics, expect takes place that in aDNA degradation exacerbated endogenous content, libraries built with this method this with built libraries content, endogenous samples in this study were amplified with the same conditions so we consider this unlikely to unlikely to this we so consider conditions same the with study amplified this in were samples Although libraries from Method H yielded more unique yielded more H Method from data. Although libraries contexts. However, significant differences exist in the composition of recovered the sequence composition the in exist However, differences significant contexts. recovering endogenous DNA in archaeological and historic skeletal samples from tropical from tropical samples skeletal historic and archaeological DNA in endogenous recovering
Meyer, 2017), are often lost during target enrichment capture (Ávila Arcos et al., 2015). et al., 2015). (Ávila Arcos capture enrichment during target lost areMeyer, often 2017), single stranded library preparation (Gansauge et al., 2017; Gansauge & Meyer, 2013; Glocke & Glocke & 2013; Gansauge Meyer, 2017; et al., (Gansauge preparation library single stranded W This study has demonstrated that Method D and Method H were similarly efficient in in efficient similarly H were D and Method Method that study has demonstrated This Author only. samples tooth restricted are to work from this derived insights the that e note also Manuscript This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology John Wiley&Sons, Inc. CONCLUSIONS CONCLUSIONS 10 also had higher clonality and yielded more and had clonality higher also Because of the Because of the fragments. ler aDNA sequence reads and higher reads sequence
preservation patterns. patterns. preservation Aird, D., Ross, M. G., Chen, W. S., Danielsson, M., Fennell, T., Russ, C., Jaffe, D. B., Nusbaum, D. B., Jaffe, C., Nusbaum, Fennell, M., T.,Russ, Danielsson, S., W. G., Chen, M. D.,Aird, Ross, Adler, C. J., Haak, W., Donlon, D., & Cooper, A. (2011). Survival and recovery of DNA from of DNA and recovery (2011). Survival D., & Donlon, A. Cooper, Haak, W., J., Adler, C. Short Read Archive (SRA) under BioProject PRJNA398385. PRJNA398385. BioProject under (SRA) Read Archive Short M.N.C. wrote the article, with input from co authors. Genetic data are available in the NCBI the in available are Genetic data from co authors. input with wrote article, the M.N.C. the experiments, M.N.C. analyzed the data, A.C.S and T.S. provided materials and resources, and resources, materials provided and T.S. data, A.C.S analyzed the M.N.C. experiments, the Allentoft, M. E., Collins, M., Harker, D., Haile, J., Oskam, C. L., Hale, M. L., Campos, P. F.,L.,L., P. Campos, M. C. Oskam, Hale, Harker, D., Haile, M., J., E.,Collins, M. Allentoft, A.C., V.T. and T.S. provided samples and taphonomic information, M.N.C. and A.O. performed and A.O. performed M.N.C. information, and taphonomic samples provided V.T. and T.S. A.C., with Gombe samples was provided by the by provided the was samples Gombe with completed. completed. BCS 1622479 and Rust Family Foundation grant in Archaeology to M.N.C. Funding work for Archaeology M.N.C. grant in to Foundation Family and Rust BCS 1622479 Completion Fellowship program for providing financial assistance during the time this work was this time the during financial assistance program for Fellowship providing Completion Andrews, R. M., Kubacka, I., Chinnery, P. F., Lightowlers, R. N., Turnbull, D. M., & Howell, N. & D. M., Howell, N.,Turnbull, R. Lightowlers, I., F., P. Chinnery, Kubacka, M., Andrews, R. BCS 061272 to W.J.P., National Science Foundation Doctoral Dissertation Improvement Award Doctoral Dissertation Foundation Science National W.J.P., to BCS 061272 Origins’ thank the Arizona State University School of Human Evolution and Social Change Dissertation Dissertation Change and Social Evolution Human of University School State Arizona the thank relatively well versus poorly preserved samples and/or for differences in site specific aDNA site specific in for differences and/or samples preserved versus poorly well relatively Office of the President of Arizona State University to A.C.S, and the Institute and of the Human A.C.S, to University State Arizona of President Office the of Pedro y Elena Hernández, the Selz Foundation, and Jerry Murdoch. M.N.C wishes to further to wishes M.N.C and Jerry Foundation, Murdoch. Selz the y Hernández, Pedro Elena chimpanzees. We especially thank Dr. Jane Goodall fo Goodall Jane Dr. especially thank We chimpanzees. In addition, we also thank Dr. L. Antonio Curet and Curet L. Antonio In Dr. we thank also addition, samples. the differences between for control will results study that so of settings data the sub forallow finer
material and making them available for further available study for them andmaterial making this paper. this Wilson, Dr. Anne Pusey, and Dr. Ian Gilby forIan facili Gilby and Dr. Dr. Anne Pusey, Wilson, Yaxuna, which was generously supported by the Fundación Roberto Hernández, Fundación Hernández, Roberto Fundación the by supported generously was which Yaxuna, we thank the three reviewers whose thoughtful commen whose thoughtful reviewers we three the thank Instituto Nacional de Antropología e Historia for granting the permits to conduct the research at research the conduct to granting for permits e the Historia de Antropología Nacional Instituto Archaeological Park, and the Yaxuna archaeological site for invaluable logistics support. support. for logistics invaluable site archaeological Yaxuna the and Park, Archaeological discussion, and the support staff and volunteers at the Ceremonial Center of Tibes Tibes Center of Ceremonial the at staff and volunteers and support the discussion, N., & Bunce, M. (2012). The half life of DNA in bone: measuring decay kinetics in 158 158 in kinetics decay measuring bone: in of DNA The half life (2012). M. Bunce, N., & C., & Gnirke, A. (2011). Analyzing and minimizing PCR amplification bias in Illumina in bias amplification PCR and minimizing Analyzing (2011). &C., Gnirke, A. ancient teeth and bones. bones. ancient and teeth
Samaniego, J. A., Gilbert, M. T., Willerslev, E., Zhang, G., Scofield, R. P., Holdaway, R. Holdaway, R. P., R. E.,Zhang,G., Scofield, T.,Willerslev, M. A., Gilbert, Samaniego, J. sequencing libraries. libraries. sequencing Author contributions were as follows: M.N.C. and A.C.S. designed the research, W.J.P, research, designed W.J.P, the A.C.S. and M.N.C. as were follows: contributions Author (1999). Reanalysis and revision of the Cambridge reference sequence for human for human reference sequence of Cambridge the revision and (1999). Reanalysis (1748), 4724 4733. (1748), 4724 4733. 279 Sci, Biol Proc dated fossils. The authors would like to thank the research team at research the thank team to like The would authors Research with Puerto Rico samples was supported by National Science Foundation Grant Foundation Science by National was supported samples Rico Puerto with Research mitochondrial DNA. mitochondrial
project. We thank the Consejo de Arqueología of the de of Arqueología Consejo the thank project. ASU We at Origins Human and DNA Author Manuscript This article isprotected by copyright. All rights reserved. Nature Genetics, 23 Nature (2), R18. (2), 12 R18. Biology, Genome American JournalofPhysicalAnthropology Journal of Archaeological Science, 38 Archaeological of Journal ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS LITERATURE CITED LITERATURE John Wiley&Sons, Inc. , ASU Strategic Initiative Funds, Initiative Strategic , ASU Foundation Leakey 11 (2), 147 147. (2), 147 147. tating research with the Gombe Gombe tating the research with , and Dr. Rebecca Nockerts for preparing for preparing Nockerts Dr. Rebecca , and r ensuring the preservation of skeletal of skeletal preservation the r ensuring ts and suggestions led to improvement of improvement to led and suggestions ts the Gombe National Park, Dr. Mike Dr. Mike Park, National Gombe the Dr. Tim Webster for helpful Webster Dr. Tim (5), 956 964. 956 964. (5), Lastly, Page 12of24
Page 13of24
Ávila Arcos, M. C., Sandoval Velasco, M., Schroeder, H., Carpenter, M. L., Malaspinas, A. S., A. S., L., Malaspinas, M. H., Carpenter, Schroeder, M., Sandoval Velasco, Ávila Arcos, C., M. Auguie, B. (2016). gridExtra: Miscellaneous Functions for "Grid" Graphics: R package version package R version for "Grid" Graphics: Functions Miscellaneous gridExtra: B.Auguie, (2016).
Briggs, A. W., Stenzel, U., Johnson, Philip L. F., Green, R. E., Kelso, J., M., Prüfer, J., K., Meyer, L. Green, Philip E.,Kelso, F., R. U., Johnson, Stenzel, Briggs, A. W., Barlow, A., Gonzalez Fortes, G. M., Dalen, L., Pinhasi, R., Gasparyan, B., Rabeder,B., G., Gasparyan, R., L., Dalen, G. Pinhasi, M., Fortes, A., Gonzalez Barlow, Brown, J. H. (2014). Why are there so many species in the tropics? tropics? the in species many so there H. (2014).are WhyBrown, J. Buzas, M. A., Collins, L. S., & Culver, S. J. (2002). Latitudinal difference in biodiversity caused caused biodiversity (2002). differenceLatitudinal in L. J. & S. S., Culver, A., Collins, M. Buzas, Boessenkool, S., Hanghoj, K., Nistelberger, H. M., Der Sarkissian, C., Gondek, A. T.,Orlando, Gondek, C., DerH. M., Sarkissian, K., Nistelberger, Hanghoj, Boessenkool, S., Dabney, J., Knapp, M., Glocke, I., Gansauge, M. T., Weihmann, A., Nickel, B., Valdiosera, C., C., A., B., Nickel, Valdiosera, T.,Weihmann, I., M. Glocke, Knapp, M., Gansauge, Dabney, J., Cooper, A., & Poinar, H. (2000). Ancient DNA: Do It Right or Not at All. Science, 289 It at All. or Not Do Right (2000). DNA: Ancient H. A., &Cooper, Poinar, Dabney, J., & Meyer, M. (2012). Length and GC biases during sequencing library amplification: library amplification: sequencing during GC biases Length and (2012). & M. Meyer, Dabney, J., Carpenter, M. L., Buenrostro, J. D., Valdiosera, C., Schroeder, H., Allentoft, M. E., Sikora, M., M., E.,Sikora, M. H., Allentoft, Schroeder, D., Valdiosera, C., J. Buenrostro, L.,Carpenter, M. Cold Spring Harbor Harbor Spring Cold DNA damage. Ancient & (2013b). S. Pääbo, Meyer, M., Dabney, J., Fu, Q., Mittnik, A., Johnson, P. L., Bos, K., Lari, M., Bollongino, R., Sun, C., Giemsch, L., Giemsch, C., Sun, L., Bollongino,Lari, R., M., K., P. Bos, A., Johnson, Fu, Q., Mittnik, Damgaard, P. B., Margaryan, A., Schroeder, H., Orlando, L., Willerslev, E., & Allentoft, M. E. M. E.,&L., Allentoft, Willerslev, H., Orlando, A., Schroeder, B., Margaryan, Damgaard, P. Daley, T., & Smith, A. D. (2013). Predicting the molecular complexity of sequencing libraries. libraries. sequencing of molecular complexity the A. D. (2013). Predicting & Smith, Daley, T., by higher tropical rate of increase. increase. of rate tropical by higher Reports, 5 Reports, Nature Methods, 10 Methods, Nature Perspectives in Biology, 5 Biology, Perspectives in 2.2.1. Retrieved from https://cran.r project.org/package=gridExtra from https://cran.r project.org/package=gridExtra Retrieved 2.2.1. Wales, N., Peñaloza, F., Bustamante, C. D., Gilbert, M. T. P., & Bunce, M. (2015). & T.P., M. Bunce, M. D., Gilbert, Bustamante, C. F., N.,Peñaloza, Wales, Comparative performance of two whole genome capture methodologies on ancient DNA on methodologies capture whole genome of two performance Comparative genomic DNA sequences from a Neandertal. from a Neandertal. DNA sequences genomic Krause, J., Ronan, M. T., Lachmann, M., & Pääbo, S. (2007). Patterns of damage in in of damage (2007). Patterns S. &Lachmann, Pääbo, T., M., M. Ronan, Krause, J., Frischchauf, C., Paijmans, J. L. A., & Hofreiter, M. (2016). Massive influence of DNA of influence L. (2016). Massive M. & J. Hofreiter, A., Paijmans, Frischchauf, C., Illumina libraries. ancient DNA recovery from bones. bones. from recovery ancient DNA sequencing libraries. libraries. sequencing 41 L., Barrett, J. H., & Star, B. (2016). Combining bleach and mild predigestion improves improves predigestion and mild bleach B. H., (2016). & Combining Star, Barrett,L., J. Pulling out the 1%: whole genome capture for the targeted enrichment of ancient DNA ancient of targeted for enrichment capture the whole genome 1%: the out Pulling Sciences, 104 Wang, J., Gilbert, M. T., Willerslev, E., Greenleaf, W. J., & Bustamante, C. D. (2013). & Bustamante, C. W. J., E.,Greenleaf, T.,Willerslev, M. Gilbert, Wang, J., isolation and library preparation approaches on palaeogenomic sequencing data. sequencing palaeogenomic on approaches and library preparation isolation Theodossiev, N., Pettener, D., Luiselli, D., Sandoval, K., Moreno Estrada, A., Li,A., Y., K., Moreno Estrada, D.,Luiselli, Sandoval, D., N.,Pettener, Theodossiev, Garcia, N., Paabo, S., Arsuaga, J. L., & Meyer, M. (2013a). Complete mitochondrial L., mitochondrial (2013a).& M. Complete Meyer, J. Arsuaga, S., N., Paabo, Garcia, 1139. 1139. a comparison of various polymerase buffer systems with ancient DNA and modern with systems polymerase buffer ofa various comparison Rasmussen, M., Gravel, S., Guillen, S., Nekhrizov, G., Leshtakov, K., Dimitrova, D., Leshtakov, K., Nekhrizov, Dimitrova, G., S., Guillen, S., Gravel, M., Rasmussen, 99 Proceedings of the National Academy of Sciences,of 110 Academy National the of fragments. Proceedings genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA from ultrashort reconstructed bear cave Pleistocene a Middle of sequence genome sequencing libraries. libraries. sequencing Schmitz, R., Burger, J., Ronchitelli, A. M., Martini, F., Cremonesi, R. G., Svoboda, J., J., Svoboda, G., R. F., Martini, Cremonesi, A. M., Ronchitelli, Burger,R., J., Schmitz, Scientific Scientific and teeth. bones ancient DNA in endogenous to Improving access (2015). Bauer, P., Caramelli, D., Castellano, S., Reich, D., Paabo, S., & Krause, J. (2013). A & J. D., S., Reich, Paabo, Krause, S., Castellano, D., Caramelli, Bauer, P., (1), 8 22. (1), 8 22. Author Manuscript (12), 7841–7843. , 11184. , 11184. (37), 14616–14621. (37), 14616–14621. This article isprotected by copyright. All rights reserved. Methods in Ecology and Evolution, 6 Evolution, Ecology and in Methods (4), 325 327. (4), 325 327. (5), 852 864. 852 864. Genetics,(5), 93 Human of Journal American (2), 87 94. (2), 52 87 94. BioTechniques, American JournalofPhysicalAnthropology (7), a012567. (7), a012567. John Wiley&Sons, Inc. Proceedings of the National Academy of Sciences, Academy of National the of Proceedings Molecular Ecology Resources Ecology Molecular 12 Proceedings of the National Academy of National the of Proceedings (6), 725 734. (6), 725 734. Journal of Biogeography, Journal Biogeography, of . . (39), 15758 15763. 15758 15763. (39), (5482), bioRxiv . .
Gutierrez Garcia, T. A., Vazquez Dominguez, E., Arroyo Cabrales, J., Kuch, M., Enk, J., King, J., Enk, Kuch, M., J., Arroyo Cabrales, E., Vazquez Dominguez, A., Gutierrez Garcia, T. Head, S. R., Komori, H. K., LaMere, S. A., Whisenant, T., Van Nieuwerburgh, F., Salomon, D. F., Salomon, T.,Van Nieuwerburgh, LaMere, A., Whisenant, S. K., H. Komori, Head, R., S. Günther, T., Valdiosera, C., Malmstrom, H., Ureña, I., Rodriguez Varela, R., Sverrisdóttir, Ó. Sverrisdóttir, R., I., H., Ureña, Rodriguez Varela, Malmstrom, C., Günther, T.,Valdiosera, Glocke, I., & Meyer, M. (2017). Extending the spectrum of DNA sequences retrieved from retrieved sequences of DNA spectrum the (2017). Extending I., & M. Meyer, Glocke, Heintzman, P. D., Zazula, G. D., Cahill, J. A., Reyes, A. V., MacPhee, R. D., & Shapiro, B. & Shapiro, A., Reyes, D., R. A. J. V., MacPhee, G. D., Cahill, D., Zazula, P. Heintzman, (1), 1 11. (1), 10 1 11. Mitochondrion, mitogenomics. T. (2010). Ancient Y., & M. S. Ho, Gilbert, Ginolhac, A., Rasmussen, M., Gilbert, M. T. P., Willerslev, E., & Orlando, L. (2011). E.,& Willerslev, Orlando, T.P., M. Gilbert, M., A., Rasmussen, Ginolhac, Hofreiter, M., Paijmans, J. L., Goodchild, H., Speller, C. F., Barlow, A., Fortes, G. G., Thomas, G. G., Thomas, Fortes, Barlow, F., L., A., C. H., Speller, J. Goodchild, Hofreiter, Paijmans, M., Gansauge, M. T., & Meyer, M. (2013). Single stranded DNA library preparation for the preparation DNA library (2013). Single stranded M. &Gansauge, Meyer, M. T., Gilbert, M. T. P., Hansen, A. J., Willerslev, E., Turner Walker, G., & Collins, M. (2006). M. & Collins, G., E.,Turner Walker, Willerslev, A. J., Hansen, T.P., M. Gilbert, Gansauge, M. T., Gerber, T., Glocke, I., Korlević, P., Lippik, L., Nagel, S., Riehl, L. Riehl, M., Lippik,L., Nagel, S., P., I., T.,Glocke, Korlević, Gerber, Gansauge, M. T., Gamba, C., Jones, E. R., Teasdale, M. D., McLaughlin, R. L., Gonzalez Fortes, G., Mattiangeli, G., Mattiangeli, L.,Gonzalez Fortes, R. D., McLaughlin, Teasdale, E.R., M. Jones, Gamba, C.,
Ghasemi, A., & Zahediasl, S. (2012). Normality tests for statistical analysis: a guide for a guide non analysis: for statistical (2012). Normality tests S. Zahediasl, & A., Ghasemi, Gamba, C., Hanghoj, K., Gaunitz, C., Alfarhan, A. H., Alquraishi, S. A., Al Rasheid, K. A., A., Al Rasheid, S. H., Alquraishi, Alfarhan, C., A. Gaunitz, K., Hanghoj, Gamba, C., (7), 553 559. (7), 23 553 559. Biology, C., & Poinar, H. N. (2014). Ancient DNA and the tropics: a tale. rodent's tropics: and the DNA Ancient & H. N.(2014). C., Poinar, Sciences, 112 Atapuerca in Spain to modern day Basques. Basques. modern day to Spain Atapuerca in 10 Götherström, A., & Jakobsson, M. (2015). Ancient genomes link early farmers from farmers genomes early link (2015). Ancient M. & Jakobsson, A., Götherström, M., Carbonell, E., Dunn, M., Storå, J., Iriarte, E., Arsuaga, J. L., Carretero, J. M., J. M., L., Iriarte, Arsuaga, Carretero, J., J. Storå, M., E., E.,Dunn, Carbonell, M., O., Daskalaki, E. A., Skoglund, P., Naidoo, T., Svensson, E. M., Bermudez de Castro, J. J. de Castro, Bermudez E.M., T.,Svensson, Naidoo, P., O., Daskalaki, E.A., Skoglund, overviews and challenges. challenges. overviews and ancient bones and teeth. andancient teeth. bones 27 R., & Ordoukhanian, P. (2014). Library construction for next generation sequencing: sequencing: for next generation Libraryconstruction (2014). P. &R., Ordoukhanian, Evolutionary History. Evolutionary History. (2015). Genomic Data from Extinct North American Camelops Revise Camel Camel Revise American North Camelops Extinct from Data (2015). Genomic Bioinformatics, DNA Bioinformatics, sequences. ancient in damage patterns for mapDamage: testing 16 International Journal of Osteoarchaeology, Osteoarchaeology, of Journal International sources of DNA. exogenous with samples J. A., Ludwig, A., & Collins, M. J. (2015). The future of ancient DNA: Technical Technical DNA: (2015). The future of ancient J. M. A., Ludwig,&J. Collins, A., sequencing of ancient or damaged DNA. damaged DNA. ancient or ofsequencing Insights into the processes behind the contamination of degraded human teeth and bone teeth human of degraded contamination the behind processes the Insights into Nature Communications, 5 Communications, Nature prehistory. transect of European a five in millennium stasis (10), e79 e79. e79 e79. Research,(10), 45 Acids DNA Nucleic ligase. T4 DNA using degraded Higham, T. F., Hofreiter, M., Bradley, D. G., & Pinhasi, R. (2014). Genome flux and flux (2014). Genome R. & Pinhasi, D. Bradley, G., M., F.,Higham, Hofreiter, T. Schmidt, A., & Meyer, M. (2017). Single stranded DNA library preparation from highly preparation DNA library (2017). Single stranded A., &M. Meyer, Schmidt, V., Domboroczki, L., Kovari, I., Pap, I., Anders, A., Whittle, A., Dani, J., Raczky, A., Dani, J., P., A., Whittle, I.,L.,I., Anders, Pap, Kovari, V., Domboroczki, 5257. 5257. 16 statisticians. statisticians. revised timescale for human evolution based on ancient mitochondrial genomes. genomes. mitochondrial based ancient on evolution for human revised timescale advances and conceptual shifts. shifts. and conceptual advances extraction methods for high throughput sequencing. for sequencing. high throughput methods extraction Bradley, D. G., & Orlando, L. (2016). Comparing the performance of three ancient DNA three the performance of L. Comparing (2016). & Orlando, D.Bradley, G., (6). (6). (15), 2153 2155. (15), 2153 2155. (2), 156 164. (2), 156 164. Author Manuscript (2), 459 469. Internaiontal Journal of Endocrinology and Metabolism, 10 Metabolism, and Endocrinology of Journal Internaiontal (38), 1917–11922. (38), 1917–11922. This article isprotected by copyright. All rights reserved. Molecular Biology and Evolution, 32 Evolution, and Biology Molecular American JournalofPhysicalAnthropology Genome Research, 27 Genome Research, (2), 61 64, 66, 68, passim. passim. 68, 66, 61 64, (2), 56 BioTechniques, John Wiley&Sons, Inc. Bioessays, 37 Bioessays, 13 (4), 737 748. 8(4), 737 748. Protocols, Nature Proceedings of the National Academy of National the of Proceedings (3), 284 293. 284 293. (3), (7), 1230 1237. 1230 1237. (7), Molecular Ecology Resources, Ecology Resources, Molecular (9), 2433 2440. (9), 2433 2440. (2), 486 489. 486 489. (2), Biology Letters, Letters, Biology Current Current , Page 14of24
Page 15of24
Migration, admixture and genetic and admixture (2016). Migration, C. A. & Stone, J., A., Benn Torres, M. Nieves Colon, Meyer, M., Fu, Q., Aximu Petri, A., Glocke, I., Nickel, B., Arsuaga, J. L., Martinez, I.,L., Gracia, Martinez, J. Arsuaga, I., B., Nickel, A., Glocke, Fu, Q., Aximu Petri, Meyer, M., Meyer, M., Arsuaga, J. L., de Filippo, C., Nagel, S., Aximu Petri, A., Nickel, B., Martinez, I., A., B., Nickel, Martinez, Aximu Petri, L., Nagel, S., C., de Filippo, J. Meyer, Arsuaga, M., Lindahl, T. (1993). Instability and decay of the primary structure of DNA. of structure of primary the and decay Instability Lindahl, T.(1993). Korlevic, P., Gerber, T., Gansauge, M. T., Hajdinjak, M., Nagel, S., Aximu Petri, A., & Meyer, Aximu Petri, Nagel, S., M., Hajdinjak, Gansauge, T., M. Gerber, T., Korlevic, P., Jónsson, H., Gonolhac, A., Schubert, M., Johnson, P., & Orlando, L. (2013). mapDamage2.0: mapDamage2.0: (2013). L. & P., Orlando, Johnson, H., M., Gonolhac, A., Schubert, Jónsson, Meyer, M., & Kircher, M. (2010). Illumina sequencing library preparation for highly multiplexed for highly multiplexed preparation Illumina sequencing library (2010). & M. Kircher, Meyer, M., Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with Burrows–Wheeler Burrows–Wheeler alignment with read accurate short Fast and (2009). &Li, R. Durbin, H., Krause, J., Briggs, A. W., Kircher, M., Maricic, T., Zwyns, N., Derevianko, A., & S. Pääbo, Derevianko, Zwyns, N., T., Kircher, Briggs, Maricic, A. M., W., Krause, J., Mendisco, F., Pemonge, M. H., Leblay, E., Romon, T., Richard, G., Courtaud, P., & Deguilloux, & P., Deguilloux, Leblay, T., G., Richard, Courtaud, H., E.,Romon, M. F.,Mendisco, Pemonge, Kistler, L., Ware, R., Smith, O., Collins, M., & Allaby, R. G. (2017). A new model for ancient for ancient model & A M., new Allaby, G. (2017). R. O., Collins, L., Smith, R., Kistler, Ware, Kehlmaier, C., Barlow, A., Hastings, A. K., Vamberger, M., Paijmans, J. L., Steadman, D. W., D. W., L., Steadman, J. Paijmans, K., M., Vamberger, A., Hastings, A. Barlow, Kehlmaier, C., Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., G., Abecasis, Homer, N.,Marth, J., T.,Ruan, A., Fennell, B.,Li, Wysoker, H., Handsaker, Kircher, M., Sawyer, S., & Meyer, M. (2012). Double indexing overcomes inaccuracies in inaccuracies in overcomes indexing Double (2012). & M. Meyer, S., Kircher, Sawyer, M., Höss, M., & Pääbo, S. (1993). DNA extraction from Pleistocene bones by a silica based a by bones Pleistocene from DNA extraction & (1993). M., S. Höss, Pääbo, Nature DNA. Nature (2001). Ancient S. Pääbo, & H., Kuch, M., Poinar, D., Hofreiter, Serre, M.,
purification method. method. purification . 2. Genetics, Reviews A., de Castro, J. M., Carbonell, E., & Paabo, S. (2014). A mitochondrial genome genome A mitochondrial E.,& (2014). Carbonell, S. Paabo, M., J. A., de Castro, Nature, 531 Nature, hominins. Huesos Paabo, S. (2016). Nuclear DNA sequences from the Middle Pleistocene Sima de los de los Sima Pleistocene Middle from the DNA sequences (2016). Nuclear S. Paabo, Gracia, A., Bermudez de Castro, J. M., Carbonell, E., Viola, B., Kelso, J., Prufer,K., & J., Carbonell, M., B.,E., Viola, Kelso, J. Castro, de A., Bermudez Gracia, sequence of a hominin from Sima de los Huesos. Huesos. de los Sima from asequence hominin of ancient bones and teeth. andancient teeth. bones of the Royal Society 284 B, Royal the of Sciences, 370 M. (2015). Reducing microbial and human contamination in DNA extractions from DNA extractions in contamination and human microbial (2015). Reducing M. (3), 20 231 236. Biology, Current fast approximate Bayesian estimates of ancient DNA damage parameters. damage of parameters. ancient DNA estimates Bayesian fast approximate target capture and sequencing. and sequencing. target capture 6310 6320. 6310 6320. (14), 1754 1760. (14), 25 1754 1760. transform. Bioinformatics, in the Lesser the in Antilles. relationships of the extinct Bahamian giant tortoise Chelonoidis alburyorum. alburyorum. giant Chelonoidis tortoise Bahamian of extinct the relationships (2010). A Complete mtDNA Genome of an Early Modern Human from Kostenki, Russia. Russia. Kostenki, from Modern Human an Early Genome of mtDNA (2010). A Complete (16), 2078 2079. (16), 25 2078 2079. Bioinformatics, format and SAMtools. M. F. (2015). Where are the Caribs? Ancient DNA from ceramic period human remains remains human DNA Ancient period from ceramic Caribs? the are F.M. (2015). Where DNA decay based on paleogenomic meta analysis. meta analysis. paleogenomic on based DNA decay Albury, N. A., Franz, R., Hofreiter, M., & Fritz, U. (2017). Tropical ancient DNA reveals DNA reveals ancient U. (2017). Tropical Fritz, & Hofreiter, R., M., N. A., Franz, Albury, 29 Durbin, R., & Genome Project Data Processing, S. (2009). The Sequence Alignment/Map Alignment/Map (2009). The Sequence S. Data Project Processing, & R., Genome Durbin, multiplex sequencing on the Illumina platform. Illumina the platform. sequencing on multiplex the American Association of Physical Anthropologists, New Orleans, LA. Abstract LA. New Orleans, of Physical Anthropologists, Association Americanthe continuity in pre and post-contact Puerto Rico Puerto post-contact and pre in continuity retrieved from: http://onlinelibrary.wiley.com/doi/10.1002/ajpa.23210/full. http://onlinelibrary.wiley.com/doi/10.1002/ajpa.23210/full. from: retrieved Author Manuscript (13), 1682 1684. (1660), 20130388. (1660), 20130388. This article isprotected by copyright. All rights reserved. Nucleic Acids Research, 21 Research, Acids Nucleic American JournalofPhysicalAnthropology Philosophical Transactions of the Royal Society B: Biological Society B: Royal the of Biological Transactions Philosophical (2), 87 93. (2), 59 87 93. BioTechniques, (1846). (1846). (7595), 504 507. (7595), 504 507. Cold Spring Harbor Protocols, 6 Protocols, Harbor Spring Cold John Wiley&Sons, Inc. 14 . Paper presented at the 86th Meeting of Meeting of 86th at the presented . Paper (1), e3. e3. (1), 40 Research, Acids Nucleic (16), 3913 3914. 3913 3914. (16), Nature, 505 Nature, (11), 45 Research, Acids Nucleic (7483), 403 406. (7483), 403 406. , 1 10. , 1 10. Nature, 362 Nature, Bioinformatics, Bioinformatics, Proceedings Proceedings , 709 715. , 709 715.
Pinhasi, R., Fernandes, D., Sirak, K., Novak, M., Connell, S., Alpaslan Roodenberg, S., S., Alpaslan Roodenberg, S., Connell, K., Fernandes, Novak, M., Sirak, R., D., Pinhasi, Pestle, W. J., & Colvard, M. (2012). Bone collagen preservation in the tropics: a case study from a case from tropics: the in study preservation collagen (2012). & Bone M. Colvard, W. J., Pestle, (PhD (PhD Approach. Isotopic Rico: An Puerto Prehistoric Society in and (2010). Diet W. J. Pestle, Rohland, N., & Hofreiter, M. (2007). Ancient DNA extraction from bones and teeth. from bones extraction (2007). DNA Ancient M. N.,& Hofreiter, Rohland, Paijmans, J. L., Gilbert, M. T., & Hofreiter, M. (2013). Mitogenomic analyses from ancient from ancient analyses Mitogenomic (2013). L., T.,& M. M. Hofreiter, J. Gilbert, Paijmans, Richards, M. B., Sykes, B. C., & Hedges, R. E. M. (1995). Authenticating DNA Extracted From From Extracted DNA (1995). Authenticating M. E. B. R. & C., Hedges, B., M. Richards, Sykes, Pääbo, S. (1989). Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic and molecular cloning, characterization, Extraction, DNA: (1989). Ancient S. Pääbo, Okonechnikov, K., Conesa, A., & García Alcalde, F. (2016). Qualimap 2: advanced multi advanced 2: F. (2016). Qualimap García Alcalde, A., & K., Conesa, Okonechnikov,
Orlando, L., Ginolhac, A., Zhang, G., Froese, D., Albrechtsen, A., Stiller, M., Schubert, M., M., Schubert, M., Albrechtsen, A., Stiller, D., G., Zhang, Froese, A., L., Ginolhac, Orlando, Overballe Petersen, S., Orlando, L., & Willerslev, E. (2012). Next generation sequencing offers offers sequencing E. (2012). Next generation L., & Willerslev, Orlando, S., Overballe Petersen, Schroeder, H., Ávila Arcos, M. C., Malaspinas, A. S., Poznik, G. D., Sandoval Velasco, M., Poznik, A. S., Malaspinas, C., M. H., Ávila Arcos, Schroeder, Racimo, F., Renaud, G., & Slatkin, M. (2016). Joint Estimation of Contamination, Error of and Contamination, Estimation (2016). Joint M. & F., Slatkin, G., Renaud, Racimo, Renaud, G., Slon, V., Duggan, A. T., & Kelso, J. (2015). Schmutzi: estimation of contamination of contamination estimation (2015). Schmutzi: & J. Kelso, A. T., V., Duggan, G., Slon, Renaud, R Core Team. (2016). R: A language and environment for statistical computing. URL URL computing. for statistical and environment A language Core Team. (2016). R: R (12), 3669 3673. (12), 3669 3673. 112 Academy Sciences, of Protocols, 2 Protocols, Gerritsen, F., Moiseyev, V., Gromov, A., Raczky, P., Anders, A., Pietrusewsky, M., M., Anders, A., Pietrusewsky, P., A., Raczky, V., Gromov, F., Moiseyev, Gerritsen, ancient Puerto Rico. Rico. ancient Puerto Dissertation), University of Illinois at Chicago, Chicago. Illinois Chicago. at Chicago, of University Dissertation), Wang, X., Min, J., Zazula, G. D., Seguin Orlando, A., Mortensen, C., Magnussen, K., Magnussen, A., C., Mortensen, Zazula, G. D., Seguin Orlando, J., Wang, X., Min, Molecular Phylogenetics and Evolution, 69 Evolution, and Phylogenetics DNA. Molecular Rollefson, G., Jovanovic, M., Trinhhoang, H., Bar Oz, G., Oxenham, M., Matsumura, H., Matsumura, M., G., Oxenham, H., Bar Oz, Trinhhoang, M., G., Jovanovic, Rollefson, D., Vinther, J., Dolocan, A., Stenderup, J., Velazquez, A. M., Cahill, J., Rasmussen, M., Rasmussen, M., J., Cahill, A. M., Velazquez, J., Dolocan, A., Stenderup, J., D., Vinther, Ancient Skeletal Remains. Remains. Skeletal Ancient amplification. amplification. 224. 224. Raghavan, M., Korneliussen, T., Malaspinas, A. S., Vogt, J., Szklarczyk, D., Kelstrup, C. C. Kelstrup, Szklarczyk, D., Vogt, J., A. S., T.,Malaspinas, Korneliussen, Raghavan, M., 17th century enslaved Africans from the Caribbean. Caribbean. from the Africans enslaved 17th century (2), 32 292 Bioinformatics, sequencing data. high throughput for quality control sample Cappellini, E., Petersen, B., Moltke, I., Johnson, P. L., Fumagalli, M., Vilstrup, J. T., J. L., Vilstrup, P. M., Fumagalli, I., B., Johnson, Moltke, E.,Petersen, Cappellini, Willerslev, E., Bustamante, C. D., & Gilbert, M. T. P. (2015). Genome wide ancestry of ancestry (2015). Genome wide P. D., & T. M. C. Gilbert, E.,Bustamante, Willerslev, Miller, D. C., Antczak, D. F., Bertelsen, M. F., Brunak, S., Al Rasheid, K. A., Ryder, O.,A., Ryder, K. Al Rasheid, S., Brunak, F., D. F., Antczak, M. Bertelsen, D. C., Miller, new insights into DNA degradation. DNA degradation. into new insights 294. 294. Samaniego, J. A., Haviser, J. B., Dee, M. W., Stafford, T. W., Salas, A., Orlando, L., A., Orlando, Salas, B., Stafford, T.W., Dee, W., J. M. A., Haviser, Samaniego, J. Thompson, J. F., Weinstock, J., Gregersen, K., Roed, K. H., Eisenmann, V., Rubin, C. J., C. J., V., Rubin, K. H., Gregersen, Eisenmann, F., K., J., Roed, Weinstock, J. Thompson, Pleistocene horse. Pleistocene & Hofreiter, M. (2015). Optimal Ancient DNA Yields from the Inner Ear Part of the InnerPart Ear from the DNA Ancient Yields Optimal (2015). & M. Hofreiter, Carpenter, M. L., Moreno Mayar, J. V., Sikora, M., Johnson, P. L. E., M. P. F., Johnson, Allentoft, M., V., Sikora, J. L.,Carpenter, Moreno Mayar, M. E. (2013). Recalibrating Equus evolution using the genome sequence of an early Middle early Middle an using of the sequence genome evolution Equus E. (2013). Recalibrating Demography for Nuclear DNA from Ancient Humans. Humans. DNA from Ancient Nuclear Demography for , 16 Biology, Genome ancient DNA. for calling consensus mitochondrial and endogenous Jensen, L. J., Olsen, J. V., Hofreiter, M., Nielsen, R., Shapiro, B., Wang, J., & Willerslev, & B., Willerslev, Shapiro, Wang, J., R., V., L.Nielsen, Hofreiter, J. Olsen, M., Jensen, J., https://www.r project.org/. Vienna, Austria: R Foundation for Statistical Computing. Computing. for Statistical Foundation R Vienna, Austria: https://www.r project.org/. Andersson, L., Mundy, J., Krogh, A., Gilbert, M. T., Kjaer, K., Sicheritz Ponten, T., Kjaer,T., K., Sicheritz Ponten, M. Krogh, A., Gilbert, L., J., Mundy, Andersson, Author One, 10 PLoS Bone. Petrous Human Manuscript (7), 1756 1762. (7), 1756 1762. Proceedings of the National Academy of Sciences, 86 Academy Sciences, of National the of Proceedings This article isprotected by copyright. All rights reserved. Nature, 499 Nature, Journal of Archaeological Science, Archaeological 39 of Journal American JournalofPhysicalAnthropology Journal of Archaeological Science, 22 Science, Archaeological of Journal (7456), 74 78. (7456), 74 78. John Wiley&Sons, Inc. (6), e0129102. e0129102. (6), (7), 364 368. (7), 30 364 368. Anatomy, of Annals 15 (2), 404 416. (2), 404 416. Proceedings of the National National the of Proceedings PLoS Genetics, 12 PLoS (7), 2079 2090. 2079 2090. (7), (2), 291 299. (2), 291 299. , 1939 1943. , 1939 1943. (4), e1005972. (4), e1005972. Nature Nature Page 16of24
Page 17of24
Wade, L. (2015). Breaking a tropical taboo. Breaking taboo. L. a(2015). tropical Wade, Wales, N., Caroe, C., Sandoval Velasco, M., Gamba, C., Barnett, R., Samaniego, J. A., Samaniego, R., J. Barnett, C., Gamba, M., Sandoval Velasco, N.,Caroe, C., Wales, Smith, C. I., Chamberlain, A. T., Riley, M. S., Cooper, A., Stringer, C. B., & Collins, M. J. J. M. &B., Collins, C. A., Cooper, Stringer, A. S., T.,Riley, I., M. C. Smith, Chamberlain, Journal of Statistical Software, Software, Statistical of Journal Package. reshape the with Data H. (2007). Reshaping Wickham, Tromp, M., Buckley, H., Geber, J., & Matisoo Smith, E. A. (2017). EDTA decalcification of decalcification E.A. (2017). EDTA &Geber, Matisoo Smith, J., H., Buckley, M., Tromp, Warinner, C., Rodrigues, J. F., Vyas, R., Trachsel, C., Shved, N., Grossmann, J., Radini, A., Radini, J., N.,Grossmann, Shved, C., F., Vyas, J. Trachsel, R., Rodrigues, C., Warinner, Wickham, H. (2016). tidyr: Easily Tidy Data with `spread()` and `gather()` Functions. R package package R Functions. `gather()` and `spread()` with Data Tidy H. (2016). tidyr: Easily Wickham, Wickham, H. (2009). Wickham, Simbolo, M., Gottardi, M., Corbo, V., Fassan, M., Mafficini, A., Malpeli, G., Lawlor, R. T., &Lawlor, T., G., R. A., Malpeli, Mafficini, V., Fassan, Gottardi, M., Corbo, M., M., Simbolo, Seguin Orlando, A., Korneliussen, T. S., Sikora, M., Malaspinas, A. S., Manica, A., Moltke, I., A., Moltke, Manica, A. S., Malaspinas, M., Sikora, T.S., A., Korneliussen, Seguin Orlando, Schuenemann, V. J., Bos, K., DeWitte, S., Schmedes, S., Jamieson, J., Mittnik, A., Forrest, S., Mittnik, S., A., Forrest, J., Jamieson, S., Schmedes, S., K., DeWitte, Bos, V. J., Schuenemann, Schubert, M., Ermini, L., Der Sarkissian, C., Jonsson, H., Ginolhac, A., Schaefer, R., Martin, M. M. Martin, R., A., H., Schaefer, Ginolhac, L., Jonsson, C., Der Sarkissian, Ermini, M., Schubert,
Seguin Orlando, A., Hoover, C. A., Vasiliev, S. K., Ovodov, N. D., Shapiro, B., A., Cooper, N.D., Shapiro, Ovodov, K., S. A., Vasiliev, C. A., Hoover, Seguin Orlando, (4), 336 344. Genetics,(4), 46 336 344. Nature back at least 36,200 years. 36,200 back at least Nielsen, R., Orlando, L., & Willerslev, E. (2014). Genomic structure in Europeans dating dating Europeans structureGenomic in E.(2014). &L., Willerslev, Orlando, R., Nielsen, (2001). Not just old but old and cold? and cold? old but old just (2001). Not Journal of Archaeological Science: Reports, 14 Reports, Science: Archaeological of Journal samples. U., Salazar Garcia, D. C., Eppler, E., Seiler, R., Hansen, L. H., Castruita, J. A., Barkow L. J. H., Castruita, Hansen, R., Eppler, E.,Seiler, D.U., C., Salazar Garcia, dental calculus as an alternate means of microparticle extraction from archaeological from archaeological extraction microparticle of means an alternate calculus asdental Hancock, Y., Tito, R. Y., Fiddyment, S., Speller, C., Hendy, J., Charlton, S., Luder, H. S., Charlton, Hendy, C., Speller, S., J., Y., Fiddyment, R. Y., Tito, Hancock, (6), 59 BioTechniques, ancient DNA. for library preparation DNA versus double stranded 368 371. 368 371. Madrigal, J. R., Orlando, L., & Gilbert, M. T. (2015). New insights on single stranded single stranded on L., New insights & Orlando, T.(2015). M. R., Gilbert, J. Madrigal, & Cappellini, E. (2014). Pathogens and host immunity in the ancient human oral cavity. human ancient the in immunity and host Pathogens E.(2014). & Cappellini, Willerslev, E., von Mering, C., Lewis, C. M., Jr., Collins, M. J., Gilbert, M. T., Ruhli, F., M. Gilbert, T.,Ruhli, M. J., Collins, Lewis, Jr., M., Mering, C. E.,von C., Willerslev, Histopathological Samples. Samples. Histopathological version 0.6.0. 0.6.0. version Oesterreicher, S., Teoh, K. Y., Kelstrup, C. D., Olsen, J. V., Nanni, P., Kawai, T., P., V., Nanni, J. D., Olsen, C. K. Y., Kelstrup, Teoh, S., Oesterreicher, Scarpa, A. (2013). DNA Qualification Workflow for Next Generation Sequencing of Generation Sequencing Next for Workflow Qualification (2013). DNA Scarpa, A. 21 Lambert, D., Khartanovich, V., Wall, J. D., Nigst, P. R., Foley, R. A., Lahr, M. M., M., M. Lahr, Foley, R., D., Nigst, P. A., R. J. V., Wall, Lambert, D., Khartanovich, (2011). Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of plasmid yielding pPCP1 the of ancient pathogens enrichment (2011). Targeted Albrechtsen, A., Ko, A., Margaryan, A., Moiseyev, V., Goebel, T., Westaway, M., M., V., Goebel, T.,Westaway, A., Moiseyev, Margaryan, A.,Ko, Albrechtsen, A., Coombes, B. K., Wood, J. W., Earn, D. J. D., White, W., Krause, J., & H. N. Krause, Poinar, J., W., D., White, Earn, D. J. W., J. B. K., Wood, Coombes, 9. 9. and metagenomic analysis using PALEOMIX. using PALEOMIX. analysis and metagenomic Characterization of ancient and modern genomes by SNP detection and phylogenomic andby phylogenomic detection genomes SNP and modern of ancient Characterization Yersinia pestis from victims of the Black Death. of Black the from victims Yersinia pestis methylation patterns. patterns. methylation D., Fernandez, R., Kircher, M., McCue, M., Willerslev, E., & Orlando, L. (2014). Orlando, E.,& Willerslev, M., McCue, M., Kircher, R., D., Fernandez, DNA libraries with AccuPrime Pfx: consequences on nucleotide misincorporation and consequences misincorporation nucleotide on Pfx: AccuPrime with DNA libraries Rubin, E. M., Willerslev, E., & Orlando, L. (2015). Amplification of TruSeq ancient of TruSeq L. Amplification (2015). E.,& Willerslev, E.M., Orlando, Rubin, Sciences, 108 Author (12), 20. Manuscript (38), E746–E752. (38), E746–E752. ggplot2: Elegant Graphics for Data Analysis Data for Graphics Elegant ggplot2: This article isprotected by copyright. All rights reserved. STAR: Science & Technology of Archaeological Research, 1 Research, Archaeological & of Science Technology STAR: American JournalofPhysicalAnthropology Science, 346 PLoS One, 8 PLoS John Wiley&Sons, Inc. (6246), 370. (6246), 370. 349 Science, Nature, 410 Nature, (6213), 1113 1118. (6213), 1113 1118. 16 (6), e62692. e62692. (6), (5), 1056 1082. 9(5), 1056 1082. Protocols, Nature Proceedings of the National Academy of National the of Proceedings , 771 772. , 771 772. , 462 466. , 462 466. . New York: Springer Verlag. . New Springer Verlag. York: (1), 1
present the study. in included samples Table Skeletal 1. 3 2 1 AD 377 Yaxuna Yaxuna AD377 Yaxuna AD376 Yaxuna AD375 Yaxuna AD373 Yaxuna AD372 Yaxuna AD368 Sample Site Site Sample PC E24 E24 PC PC 117 117 PC PI 388 PI Date of individual death Date of individual calAD. probability date Radiocarbon median context. archaeological on date, based Approximate T 251 Tibes Tibes 251 T GB 7 GB 67 PI Candelero Candelero Candelero Candelero National National Paso del del Paso Paso del del Paso Gombe Gombe Punta Punta Punta Punta Indio Indio Indio Indio Author Park Manuscript Ponce, Puerto Puerto Ponce, Puerto Rico Rico Puerto Puerto Rico Rico Puerto Puerto Rico Rico Puerto Puerto Rico Rico Puerto Vega Baja, Baja, Vega Vega Baja, Baja, Vega Humacao, Humacao, Humacao, Humacao, Tanzania Tanzania Country Country Yucatán, Yucatán, Yucatán, Yucatán, Yucatán, Yucatán, Yucatán, Kigoma, Kigoma, Region, Region, México México México México México México México This article isprotected by copyright. All rights reserved. . Rico Rico American JournalofPhysicalAnthropology rpclmnon 400-600 monsoon Tropical rpclmnon 400-600 monsoon Tropical rpclmnon 1022 monsoon Tropical rpclmnon 822 monsoon Tropical rpclmnon 616 monsoon Tropical Tropical savanna savanna Tropical savanna Tropical savanna Tropical savanna Tropical savanna Tropical savanna Tropical rpclsvna 1966 savanna Tropical Köppen-Geiger Köppen-Geiger John Wiley&Sons, Inc. classification classification environment environment century century century century century century Sample Sample (A.D.) (A.D.) Age Age 6 6 6 6 6 6 th th th th th th
2 2 2 3 Tooth Tooth Tooth Tooth Tooth Tooth Tooth 1 1 1 1 1 1 1 1
Tooth Tooth Tooth portion & portion portion & portion Petrous Petrous Petrous Petrous Petrous Petrous portion portion portion Tissue Species Species Tissue Tooth Tooth Tooth Tooth Tooth Tooth Tooth Tooth Pan troglodytes troglodytes Pan schweinfurthii Homo sapiens sapiens Homo sapiens Homo Homo sapiens sapiens Homo Homo sapiens sapiens Homo Homo sapiens sapiens Homo Homo sapiens sapiens Homo Homo sapiens Homo Homo sapiens Homo sapiens Homo Homo sapiens Homo sapiens Homo
Page 18of24
Page 19of24 bolded. P<0.05 values Significant tests. of paired Table Results 2.
Percent endogenous content content endogenous Percent Percent average GC content content GC average Percent Number of mapped, unique unique mapped, of Number Average fragment length length fragment Average Damage parameter parameter Damage Damage parameter parameter Damage Percent distinct reads reads distinct Percent Damage parameter parameter Damage DNA yields (ng/µL) yields(ng/µL) DNA Test comparison Test Percent clonality clonality Percent
Author Manuscript reads δ δ
λ D S
This article isprotected by copyright. All rights reserved. Petrous portion Wilcoxon signed ranks V 4 n/a 0.8750 0.8750 n/a 4 V 0.625 n/a ranks signed Wilcoxon 0.1250 3 = V portion Petrous n/a 0.2500 n/a portion Petrous ranks signed Wilcoxon 10 = V portion Petrous 1 = V ranks signed Wilcoxon portion Petrous ranks signed Wilcoxon 0.1250 portion Petrous n/a 0.1250 portion Petrous 0 = V n/a portion Petrous ranks signed Wilcoxon 0 = V portion Petrous ranks signed Wilcoxon portion Petrous portion Petrous American JournalofPhysicalAnthropology Tissue Tissue Tooth Wilcoxon signed ranks V = 36 n/a 0.1232 0.1232 n/a 36 = V ranks signed Wilcoxon Tooth Tooth Wilcoxon signed ranks V = 4 n/a n/a 4 = V ranks signed Wilcoxon Tooth Tooth Wilcoxon signed ranks V = 2 n/a n/a 2 = V ranks signed Wilcoxon Tooth Tooth Wilcoxon signed ranks V = 4.5 n/a n/a 4.5 = V ranks signed Wilcoxon Tooth Tooth Wilcoxon signed ranks V = 7 n/a n/a 7 = V ranks signed Wilcoxon Tooth Tooth Wilcoxon signed ranks V = 46 n/a 0.0644 0.0644 n/a 46 = V ranks signed Wilcoxon Tooth Tooth Tooth Tooth Wilcoxon signed ranks V = 42 n/a 0.1601 0.1601 n/a 42 = V ranks signed Wilcoxon Tooth Tooth Tooth Tooth Wilcoxon signed ranks V = 35 n/a 0.4922 0.4922 n/a 35 = V ranks signed Wilcoxon Tooth John Wiley&Sons, Inc. Wilcoxon signed ranks ranks signed Wilcoxon Wilcoxon signed ranks ranks signed Wilcoxon Paired test Paired T test T test T test T test T test T test
t = 0.1924 9 0.8517 0.8517 9 0.1924 = t t = 0.3852 3 0.7257 0.7257 3 0.3852 = t 0.3090 3 1.2219 = t t = 0.5300 3 0.6328 0.6328 3 0.5300 = t statistic V = 30 n/a 0.8457 0.8457 n/a 30 = V V = 6 n/a 0.8750 0.8750 n/a 6 = V Test Test
DF
P-value 0.0136 0.0136 0.0058 0.0058 0.0217 0.0217 0.0371 0.0371
AuthorFigureuniqueNumber mapped, perreads1. of samples, sample. Tooth inset (A) inzoomssampleswith to Manuscript less than 60,000 reads. (B) (B) reads. 60,000 portion lessPetrous samples.than This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology 152x203mm(300 300DPI) x John Wiley&Sons, Inc.
Page 20of24
Page 21of24
Figurecomparing Boxplots distributions (calculated 2. endogenous content number percent of (A) of as Authorunique mapped, dividedreads sampledover down percent total (B) clonality reads) and (calculatedas Manuscript This article isprotected by copyright. All rights reserved. fraction of downsampled reads that are duplicates).are downsampled of that reads fraction American JournalofPhysicalAnthropology 152x177mm(300 300DPI) x John Wiley&Sons, Inc.
assuming a sequencing efforta assuming 600million of dottedreads. line The denotesrandomlyreadsnumber the of Figure 3. Extrapolation curves for shotgun library Figureforcurvesshotgun are Extrapolation 3. two forCurves the estimation. complexity shown samples with highest number of reads: PI-67 GB-7. sampleswith highest and reads:number of PI-67 zoomed inset shows Top in results forPI-67. Extrapolation curve and confidence curveand Extrapolation in preseq intervalsestimated were and using parameters default Authorsample downsampledmillion for6.8 each and pair: forGB-7 million 5.1 forreadsPI-67. reads Manuscript This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology 177x177mm(300 300DPI) x John Wiley&Sons, Inc.
Page 22of24
Page 23of24
Figure 4. Fragment length content.Boxplots Figure and (A) Fragment fragment4. GC lengths. comparing DNA distributions of Authorcomparing Boxplots distributions(C) (B) mean lengthof content. fragment %GC Scatterplot average of Manuscript This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology versus average %GC content. %GC average versus 210x254mm(300 300DPI) x John Wiley&Sons, Inc.
default parameters and assuming a sequencing effort of 600 million reads million of 600 effort a sequencing and assuming default parameters d for PI-67. reads million and 6.8 GB-7 Extrapolation curve and confidence intervals were es were intervals confidence curve and for Extrapolation PI-67. results in zoomed shows inset Top and GB-7. PI-67 of number reads: highest with samples two the F duplicates). are that reads of downsampled as fraction clonality (calculated (B) and percent reads) down sampled total over reads divided of unique number mapped, pe of (A) comparing distributions Boxplots Figure 2. samples. reads. portion 60,000 (B) Petrous than less with samples to in zooms inset samples, (A) Tooth per sample. reads unique of mapped, Figure Number 1. content. %GC versus average length of mean fragment (C) Scatterplot of average content. %GC comparing distributions Boxplots (B) fragment lengths. Figure 4. Fragment length and GC content. (A) Boxplo (A) content. and GC length Fragment Figure 4. enotes the number of reads randomly downsampled for each sample pair: 5.1 million reads for million 5.1 for pair: sample each downsampled randomly reads of number the enotes Author Manuscript for shown are Curves complexity estimation. library for shotgun curves Extrapolation igure 3. This article isprotected by copyright. All rights reserved. American JournalofPhysicalAnthropology John Wiley&Sons, Inc. rcent endogenous content (calculated as (calculated content endogenous rcent ts comparing distributions of DNA comparingts distributions timated in preseq using preseq in using timated . The dotted line line . The dotted Page 24of