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Supporting Information
Supporting Information Boothby et al. 10.1073/pnas.1510461112 SI Text tardigrades were allowed to settle and were left overnight at Repetitive Sequences in the H. dujardini Genome. A total of 4,440 room temperature. (10.72%) of the PacBio contigs, making up 7.91% of the as- sembled sequence, do not have direct homologous matches in the DNA Extraction. Isolated specimens were transferred to 1.5-mL × Illumina assembly. These contigs may represent highly repetitive microcentrifuge tubes and centrifuged at 16,837 g for 2 min to sections of the genome that are difficult to assemble with short pellet animals. Excess liquid was removed, and tubes with pel- Illumina reads, or they may be misassemblies of the PacBio data leted specimens were dipped in liquid nitrogen and simulta- neously homogenized with plastic pestles and then left briefly to due to the inherently high rate of sequencing error (10–15%). To thaw. Freeze/thaw douncing was repeated five times to homog- try to resolve this question, we aligned the Illumina reads used to enize samples. To isolate genomic DNA, homogenized samples create our assembly to all assembled PacBio contigs using Bowtie 2. were processed with Qiagen’s DNeasy Blood and Tissue Kit Alignment of our Illumina assembly to our PacBio assembly showed ’ ∼ (catalog no. 69506) according to the manufacturer s instructions. that the average coverage of the differential PacBio contigs is 66% Following isolation, genomic DNA was ethanol-precipitated. of the average coverage across all PacBio contigs, indicating that these sequences are represented in the raw Illumina data but are Assessment of EST and Core Eukaryotic Genes Mapping Approach likely repetitive, and therefore very difficult to assemble accurately. -
May Help Explain Expression of Multiple Genes in Alzheimer's
Journal of Systems and Integrative Neuroscience Research Article ISSN: 2059-9781 Micro RNA’s (miRNA’s) may help explain expression of multiple genes in Alzheimer’s Frontal Cortex James P Bennett* and Paula M Keeney Neurodegeneration Therapeutics, Inc., 3050A Berkmar Drive, Charlottesville, VA 22901, USA Abstract MicroRNA’s (miRNA’s) are non-coding RNA’s that can regulate gene expression. miRNA’s are small (~22 nt) and can bind by complementation to mRNA’s resulting in direct degradation and/or interference with translation. We used paired-end RNAseq and small RNAseq to examine expression of mRNA’s and miRNA’s, respectively, in total RNA’s isolated from frontal cortex of Alzheimer’s disease (AD) and control (CTL) subjects. mRNA’s were aligned to the hg38 human genome using Tophat2/Bowtie2 and relative expression levels determined by Cufflinks as FPKM (fragments per kilobase of exon per million mapped reads). miRNA’s were aligned to the hg19 human genome and quantitated as number of mature known miRNA reads with miRDeep*. Using a false-discovery rate (FDR) of 10% (FDR ≤ 0.10) applied to 11,794 genes with FPKM > 2.0, we identified 55 genes upregulated in AD and 191 genes downregulated in AD. The top 145 miRNA’s with the highest mean expression were mostly under-expressed in AD (132/145 miRNAs had AD/CTL < 1.0)). AD samples had increased coefficients of variation for miRNA and mRNA expression, implying greater heterogeneity. miRTAR analysis showed that 32 (58%) of the mRNA genes upregulated in AD could be controlled by one or more of 60 miRNA’s under-expressed >1.5-fold in AD (AD/CTL < 0.67). -
Stool-Derived Eukaryotic RNA Biomarkers for Detection of High-Risk Adenomas
bioRxiv preprint doi: https://doi.org/10.1101/534412; this version posted January 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Stool-derived eukaryotic RNA biomarkers for detection of high-risk adenomas Erica Barnell1,2*, Yiming Kang2,3*, Andrew Barnell2, Katie Campbell1,2, Kimberly R. Kruse2, Elizabeth M. Wurtzler2, Malachi Griffith1,4,5,6, Aadel A. Chaudhuri3,6,7+, Obi L. Griffith1,4,5,6+ 1 McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, USA 2 Geneoscopy LLC, Division of Gastroenterology and Hepatology, St. Louis, MO, USA 3 Department of Computer Science and Engineering, Washington University, St. Louis, MO, USA 4 Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA 5 Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA 6 Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA 7 Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO, USA +Corresponding author *These authors contributed equally to this work. Abbreviations AUC area under the curve LRA low-risk adenoma ANOVA analysis of variance MRA medium-risk adenoma CRC colorectal cancer NPV negative predictive value DE differentially expressed PPV positive predictive value FC fold-change ROC receiver operating characteristic FIT fecal immunochemical test seRNA stool-derived eukaryotic RNA HRA high-risk adenoma Abstract Background and aims: Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. -
Targeted Sequencing Informs the Evaluation of Normal Karyotype Cytopenic Patients for Low-Grade Myelodysplastic Syndrome
Letters to the Editor 2422 chronic lymphocytic leukemia are mostly derived from independent clones. 13 Binder M, Muller F, Jackst A, Lechenne B, Pantic M, Bacher U et al. B-cell receptor Haematologica 2014; 99: 329–338. epitope recognition correlates with the clinical course of chronic lymphocytic 9 Sanchez ML, Almeida J, Gonzalez D, Gonzalez M, Garcia-Marcos MA, leukemia. Cancer 2011; 117: 1891–1900. Balanzategui A et al. Incidence and clinicobiologic characteristics of leukemic 14 Seiler T, Woelfle M, Yancopoulos S, Catera R, Li W, Hatzi K et al. Characteri- B-cell chronic lymphoproliferative disorders with more than one B-cell clone. zation of structurally defined epitopes recognized by monoclonal antibodies pro- Blood 2003; 102: 2994–3002. duced by chronic lymphocytic leukemia B cells. Blood 2009; 114:3615–3624. 10 Klinger M, Zheng J, Elenitoba-Johnson KS, Perkins SL, Faham M, Bahler DW. Next- generation IgVH sequencing CLL-like monoclonal B-cell lymphocytosis reveals frequent oligoclonality and ongoing hypermutation. Leukemia 2015; 30: This work is licensed under a Creative Commons Attribution- 1055–1061. NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons 11 Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. license, unless indicated otherwise in the credit line; if the material is not included under Efficient generation of monoclonal antibodies from single human B cells by single the Creative Commons license, users will need to obtain permission from the license cell RT-PCR and expression vector cloning. J Immunol Methods 2008; 329: holder to reproduce the material. -
Supporting Information
Supporting Information Gnerre et al. 10.1073/pnas.1017351108 SI Materials and Methods digested vector arms. Primary packaged Fosill libraries were Illumina Sample Prep Modifications (Brief Description). Construction transfected into E. coli DH10B and amplified by overnight of fragment libraries followed standard protocols for shearing of growth at 30 °C in 750 mL 2× YT broth containing 15 μg/μL genomic DNA, end repair, adapter ligation, and size selection. chloramphenicol. Ten micrograms of purified Fosmid DNA The enrichment PCR was performed using AccuPrime Taq DNA (Qiagen QIAfilter plasmid mega purification kit) was nicked by polymerase High Fidelity (Invitrogen) with the following cycling digestion with Nb.BbvC1. The nicks were then translated by a 45- conditions: initial denaturation for 3 min at 98 °C, followed by 10 min incubation at 0 °C with DNA polymerase I and dNTPs and cycles of denaturation for 80 s at 98 °C, annealing and extension then cleaved by digestion with nuclease S1. End-repaired frag- for 90 s at 65 °C, and a final extension for 10 min at 65 °C. ments (∼100 ng) were circularized in a 500-μL ligation reaction with T4 DNA ligase. The coligated jumping fragments were then Fosmid Jumping Methods (Brief Description, with Details in the PCR amplified by inverse PCR out of the vector, using standard Following Paragraphs). Both methods make a 40-kb insert li- Illumina paired-end enrichment primers. The PCR product was brary in a Fosmid vector (1), which is transfected into Escherichia size selected to 400–600 bp and sequenced. coli. After growth, Fosmid DNA is purified from the host. -
Application of RAD-Seq in Evolutionary Genomics of Non-Model Organisms
Application of RAD-seq in Evolutionary Genomics of Non-Model Organisms By Boryana S. Koseva Submitted to the graduate degree program in Ecology and Evolutionary Biology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Chair: John K. Kelly Stuart J. Macdonald Justin P. Blumenstiel Jamie R. Walters Mark T. Holder Date Defended: 28 April 2017 ii The dissertation committee for Boryana Koseva certifies that this is the approved version of the following dissertation: Application of RAD-seq in Evolutionary Genomics of Non-Model Organisms Chair: John K. Kelly Date Approved: 11 May 2017 iii Abstract Next generation sequencing (NGS) technologies are revolutionizing how we study genetics and evolution in the modern world. Data is generated at such a fast pace that scientists are struggling to keep up with the innovations in methodology and analytical tools. Genomes are being sequenced at an unprecedented rate, and scientists in fields that until recently found no use in learning molecular techniques are venturing into the world of high-throughput sequencing. Almost 10 years ago, a research group developed Restriction-site Associated DNA Sequencing (RAD- seq), a method that targets polymorphisms in close proximity to restriction cut sites in hundreds of samples simultaneously. The beauty of RAD-seq lies in that it is highly customizable and it does not require a reference genome, or intimate prior knowledge of the genetics of the study organism one would like to use. The most exciting part about new RAD-seq methods being developed is that their accessibility has opened the door to many non-model organisms to be used in new areas of research. -
Science Journals — AAAS
SCIENCE ADVANCES | RESEARCH ARTICLE EVOLUTIONARY BIOLOGY Copyright © 2019 The Authors, some rights reserved; Unprecedented reorganization of holocentric exclusive licensee American Association chromosomes provides insights into the enigma of for the Advancement of Science. No claim to lepidopteran chromosome evolution original U.S. Government Jason Hill1,2*, Pasi Rastas3, Emily A. Hornett4,5,6, Ramprasad Neethiraj1, Nathan Clark7, Works. Distributed 8 1 9,10 under a Creative Nathan Morehouse , Maria de la Paz Celorio-Mancera , Jofre Carnicer Cols , Commons Attribution 11 7,12 1 1 13,14 Heinrich Dircksen , Camille Meslin , Naomi Keehnen , Peter Pruisscher , Kristin Sikkink , NonCommercial 9,10 15 1 1,16 17 Maria Vives , Heiko Vogel , Christer Wiklund , Alyssa Woronik , Carol L. Boggs , License 4.0 (CC BY-NC). Sören Nylin1, Christopher W. Wheat1* Chromosome evolution presents an enigma in the mega-diverse Lepidoptera. Most species exhibit constrained chromosome evolution with nearly identical haploid chromosome counts and chromosome-level gene collinearity among species more than 140 million years divergent. However, a few species possess radically inflated chromo- somal counts due to extensive fission and fusion events. To address this enigma of constraint in the face of an Downloaded from exceptional ability to change, we investigated an unprecedented reorganization of the standard lepidopteran chromosome structure in the green-veined white butterfly (Pieris napi). We find that gene content in P. napi has been extensively rearranged in large collinear blocks, which until now have been masked by a haploid chromosome number close to the lepidopteran average. We observe that ancient chromosome ends have been maintained and collinear blocks are enriched for functionally related genes suggesting both a mechanism and a possible role for selection in determining the boundaries of these genome-wide rearrangements. -
A Class I Jumping Clone Places the HLA-G Gene Approximately 100 Kilobases from Hfa-H Within the MA-A Subregion of the Human MHC
GENOMICS lo,%%914 (19%) A Class I Jumping Clone Places the HLA-G Gene Approximately 100 Kilobases from HfA-H within the MA-A Subregion of the Human MHC HRIDAYABHIRANJAN SHUKLA,* GERALD A. GILLESPIE, t RAKESH SRIVASTAVA,$ FRANCIS COLLINS,~ AND MICHAEL J. CHORNEY~~ Departments of *Human Genetics and tlnternal Medicine, Yale University School of Medicine, New Haven, Connecticut 06570; *Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037; §Departments of Internal Medicine and Human Genetics, University of Michigan, Ann Arbor, Michigan 48109; and )IDepartments of Microbiology and Immunology and Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received August 3, 1990; revised February 18, 1991 endonucleases (most of which are sensitive to C meth- By the combination of cosmid cloning, ehromosomal ylation) and pulsed-field gel electrophoresis (PFGE) jumping, and pulsed-field gel electrophoresis (PFGE), we technology, have identified the major megabase frag- have fine-mapped the HLA-A subregion of the human ma- ments upon which reside the genes encoding the jor histocompatibility complex (MIX). Through the isola- transplantation antigens, HLA-A, -B, and -C. The tion of a class I jumping clone, the Qa-like HLA-G class I positions of additional members of the class I gene gene has been placed within 100 kb of HLA-H. The tight family, such as HLA-E (Srivastava et aZ., 1987) and physical linkage of these class I genes has been further sup- cdal2 (Ragoussis et aZ., 1989), have recently been ported by hybridizing PFGE blots with locus-specific added to the molecular map. -
IS-Seq: a Novel High Throughput Survey of in Vivo IS6110 Transposition in Multiple Mycobacterium Tuberculosis Genomes
BMC Genomics This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes BMC Genomics 2012, 13:249 doi:10.1186/1471-2164-13-249 Alejandro Reyes ([email protected]) Andrea Sandoval ([email protected]) Andrés Cubillos-Ruiz ([email protected]) Katerine E Varley ([email protected]) Iván Hernández-Neuta ([email protected]) Sofía Samper ([email protected]) Carlos Martín ([email protected]) Maria Jesús Garcia ([email protected]) Viviana Ritacco ([email protected]) Lucelly López ([email protected]) Jaime Robledo ([email protected]) María Mercedes Zambrano MMZ ([email protected]) Robi D Mitra ([email protected]) Patricia Del Portillo ([email protected]) ISSN 1471-2164 Article type Research article Submission date 3 November 2011 Acceptance date 30 May 2012 Publication date 15 June 2012 Article URL http://www.biomedcentral.com/1471-2164/13/249 Like all articles in BMC journals, this peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). Articles in BMC journals are listed in PubMed and archived at PubMed Central. For information about publishing your research in BMC journals or any BioMed Central journal, go to © 2012 Reyes et al. ; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. -
Chromosome-Scale Scaffolding of De Novo Genome Assemblies Based on Chromatin Interactions
ARTICLES Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions Joshua N Burton, Andrew Adey, Rupali P Patwardhan, Ruolan Qiu, Jacob O Kitzman & Jay Shendure Genomes assembled de novo from short reads are highly fragmented relative to the finished chromosomes of Homo sapiens and key model organisms generated by the Human Genome Project. To address this problem, we need scalable, cost-effective methods to obtain assemblies with chromosome-scale contiguity. Here we show that genome-wide chromatin interaction data sets, such as those generated by Hi-C, are a rich source of long-range information for assigning, ordering and orienting genomic sequences to chromosomes, including across centromeres. To exploit this finding, we developed an algorithm that uses Hi-C data for ultra-long-range scaffolding of de novo genome assemblies. We demonstrate the approach by combining shotgun fragment and short jump mate-pair sequences with Hi-C data to generate chromosome-scale de novo assemblies of the human, mouse and Drosophila genomes, achieving—for the human genome—98% accuracy in assigning scaffolds to chromosome groups and 99% accuracy in ordering and orienting scaffolds within chromosome groups. Hi-C data can also be used to validate chromosomal translocations in cancer genomes. The Human Genome Project defined and achieved high standards for Diverse strategies have been developed to boost the contiguity the de novo assembly of reference genomes for H. sapiens and key model of de novo genome assemblies from short reads. These include end organisms. For example, the public draft human genome, reported in sequencing of fosmid clones6, fosmid clone dilution pool sequenc- 2001, contained 90% of the euchromatic sequence with an N50 (defined ing9,10, optical mapping11–14 and genetic mapping with restriction site– as the length L at which 50% of sequence is in contigs of length ≥L) of associated DNA tags15. -
Identification of Purple Sea Urchin Telomerase RNA Using a Next-Generation Sequencing Based Approach
Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach YANG LI,1,6 JOSHUA D. PODLEVSKY,2,6 MANJA MARZ,3,6 XIAODONG QI,1 STEVE HOFFMANN,4,5 PETER F. STADLER,5 and JULIAN J.-L. CHEN1,7 1Department of Chemistry & Biochemistry and 2School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA 3Department of Bioinformatics, Friedrich Schiller University of Jena, D-07743 Jena, Germany 4LIFE Center and 5Interdisciplinary Center for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany ABSTRACT Telomerase is a ribonucleoprotein (RNP) enzyme essential for telomere maintenance and chromosome stability. While the catalytic telomerase reverse transcriptase (TERT) protein is well conserved across eukaryotes, telomerase RNA (TR) is extensively divergent in size, sequence, and structure. This diversity prohibits TR identification from many important organisms. Here we report a novel approach for TR discovery that combines in vitro TR enrichment from total RNA, next-generation sequencing, and a computational screening pipeline. With this approach, we have successfully identified TR from Strongylocentrotus purpuratus (purple sea urchin) from the phylum Echinodermata. Reconstitution of activity in vitro confirmed that this RNA is an integral component of sea urchin telomerase. Comparative phylogenetic analysis against vertebrate TR sequences revealed that the purple sea urchin TR contains vertebrate-like template-pseudoknot and H/ACA domains. While lacking a vertebrate- like CR4/5 domain, sea urchin TR has a unique central domain critical for telomerase activity. This is the first TR identified from the previously unexplored invertebrate clade and provides the first glimpse of TR evolution in the deuterostome lineage. -
Reducing Assembly Complexity of Microbial Genomes with Single-Molecule Sequencing
Reducing assembly complexity of microbial genomes with single-molecule sequencing Sergey Koren1, †, Gregory P Harhay2, Timothy PL Smith2, James L Bono2, Dayna M Harhay2, D. Scott Mcvey3, Diana Radune1, Nicholas H Bergman1, and Adam M Phillippy1 Author Affiliations 1. National Biodefense Analysis and Countermeasures Center, 110 Thomas Johnson Drive, Frederick, MD 21702 2. USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933 3. USDA, ARS, Center for Grain and Animal Health Research, Manhattan, KS 66502 † Corresponding author: [email protected] This article has been published in Genome Biology: Reducing assembly complexity of microbial genomes with single-molecule sequencing. Koren S, Harhay GP, Smith TPL, Bono JL, Harhay DM, Mcvey SD, Radune D, Bergman NH, Phillippy AM. Genome Biology 14:R101 2013. Abstract Background The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. Results To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.