A Structured Query Language and Filtering Tool for the SAM/BAM File Format
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Gene Discovery and Annotation Using LCM-454 Transcriptome Sequencing Scott J
Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Methods Gene discovery and annotation using LCM-454 transcriptome sequencing Scott J. Emrich,1,2,6 W. Brad Barbazuk,3,6 Li Li,4 and Patrick S. Schnable1,4,5,7 1Bioinformatics and Computational Biology Graduate Program, Iowa State University, Ames, Iowa 50010, USA; 2Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa 50010, USA; 3Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA; 4Interdepartmental Plant Physiology Graduate Major and Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50010, USA; 5Department of Agronomy and Center for Plant Genomics, Iowa State University, Ames, Iowa 50010, USA 454 DNA sequencing technology achieves significant throughput relative to traditional approaches. More than 261,000 ESTs were generated by 454 Life Sciences from cDNA isolated using laser capture microdissection (LCM) from the developmentally important shoot apical meristem (SAM) of maize (Zea mays L.). This single sequencing run annotated >25,000 maize genomic sequences and also captured ∼400 expressed transcripts for which homologous sequences have not yet been identified in other species. Approximately 70% of the ESTs generated in this study had not been captured during a previous EST project conducted using a cDNA library constructed from hand-dissected apex tissue that is highly enriched for SAMs. In addition, at least 30% of the 454-ESTs do not align to any of the ∼648,000 extant maize ESTs using conservative alignment criteria. These results indicate that the combination of LCM and the deep sequencing possible with 454 technology enriches for SAM transcripts not present in current EST collections. -
EMBL-EBI Powerpoint Presentation
Processing data from high-throughput sequencing experiments Simon Anders Use-cases for HTS · de-novo sequencing and assembly of small genomes · transcriptome analysis (RNA-Seq, sRNA-Seq, ...) • identifying transcripted regions • expression profiling · Resequencing to find genetic polymorphisms: • SNPs, micro-indels • CNVs · ChIP-Seq, nucleosome positions, etc. · DNA methylation studies (after bisulfite treatment) · environmental sampling (metagenomics) · reading bar codes Use cases for HTS: Bioinformatics challenges Established procedures may not be suitable. New algorithms are required for · assembly · alignment · statistical tests (counting statistics) · visualization · segmentation · ... Where does Bioconductor come in? Several steps: · Processing of the images and determining of the read sequencest • typically done by core facility with software from the manufacturer of the sequencing machine · Aligning the reads to a reference genome (or assembling the reads into a new genome) • Done with community-developed stand-alone tools. · Downstream statistical analyis. • Write your own scripts with the help of Bioconductor infrastructure. Solexa standard workflow SolexaPipeline · "Firecrest": Identifying clusters ⇨ typically 15..20 mio good clusters per lane · "Bustard": Base calling ⇨ sequence for each cluster, with Phred-like scores · "Eland": Aligning to reference Firecrest output Large tab-separated text files with one row per identified cluster, specifying · lane index and tile index · x and y coordinates of cluster on tile · for each -
An Open-Sourced Bioinformatic Pipeline for the Processing of Next-Generation Sequencing Derived Nucleotide Reads
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.050369; this version posted May 28, 2020. 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 4.0 International license. An open-sourced bioinformatic pipeline for the processing of Next-Generation Sequencing derived nucleotide reads: Identification and authentication of ancient metagenomic DNA Thomas C. Collin1, *, Konstantina Drosou2, 3, Jeremiah Daniel O’Riordan4, Tengiz Meshveliani5, Ron Pinhasi6, and Robin N. M. Feeney1 1School of Medicine, University College Dublin, Ireland 2Division of Cell Matrix Biology Regenerative Medicine, University of Manchester, United Kingdom 3Manchester Institute of Biotechnology, School of Earth and Environmental Sciences, University of Manchester, United Kingdom [email protected] 5Institute of Paleobiology and Paleoanthropology, National Museum of Georgia, Tbilisi, Georgia 6Department of Evolutionary Anthropology, University of Vienna, Austria *Corresponding Author Abstract The emerging field of ancient metagenomics adds to these Bioinformatic pipelines optimised for the processing and as- processing complexities with the need for additional steps sessment of metagenomic ancient DNA (aDNA) are needed in the separation and authentication of ancient sequences from modern sequences. Currently, there are few pipelines for studies that do not make use of high yielding DNA cap- available for the analysis of ancient metagenomic DNA ture techniques. These bioinformatic pipelines are tradition- 1 4 ally optimised for broad aDNA purposes, are contingent on (aDNA) ≠ The limited number of bioinformatic pipelines selection biases and are associated with high costs. -
Sequence Alignment/Map Format Specification
Sequence Alignment/Map Format Specification The SAM/BAM Format Specification Working Group 3 Jun 2021 The master version of this document can be found at https://github.com/samtools/hts-specs. This printing is version 53752fa from that repository, last modified on the date shown above. 1 The SAM Format Specification SAM stands for Sequence Alignment/Map format. It is a TAB-delimited text format consisting of a header section, which is optional, and an alignment section. If present, the header must be prior to the alignments. Header lines start with `@', while alignment lines do not. Each alignment line has 11 mandatory fields for essential alignment information such as mapping position, and variable number of optional fields for flexible or aligner specific information. This specification is for version 1.6 of the SAM and BAM formats. Each SAM and BAMfilemay optionally specify the version being used via the @HD VN tag. For full version history see Appendix B. Unless explicitly specified elsewhere, all fields are encoded using 7-bit US-ASCII 1 in using the POSIX / C locale. Regular expressions listed use the POSIX / IEEE Std 1003.1 extended syntax. 1.1 An example Suppose we have the following alignment with bases in lowercase clipped from the alignment. Read r001/1 and r001/2 constitute a read pair; r003 is a chimeric read; r004 represents a split alignment. Coor 12345678901234 5678901234567890123456789012345 ref AGCATGTTAGATAA**GATAGCTGTGCTAGTAGGCAGTCAGCGCCAT +r001/1 TTAGATAAAGGATA*CTG +r002 aaaAGATAA*GGATA +r003 gcctaAGCTAA +r004 ATAGCT..............TCAGC -r003 ttagctTAGGC -r001/2 CAGCGGCAT The corresponding SAM format is:2 1Charset ANSI X3.4-1968 as defined in RFC1345. -
Multi-Scale Analysis and Clustering of Co-Expression Networks
Multi-scale analysis and clustering of co-expression networks Nuno R. Nen´e∗1 1Department of Genetics, University of Cambridge, Cambridge, UK September 14, 2018 Abstract 1 Introduction The increasing capacity of high-throughput genomic With the advent of technologies allowing the collec- technologies for generating time-course data has tion of time-series in cell biology, the rich structure stimulated a rich debate on the most appropriate of the paths that cells take in expression space be- methods to highlight crucial aspects of data struc- came amenable to processing. Several methodologies ture. In this work, we address the problem of sparse have been crucial to carefully organize the wealth of co-expression network representation of several time- information generated by experiments [1], including course stress responses in Saccharomyces cerevisiae. network-based approaches which constitute an excel- We quantify the information preserved from the origi- lent and flexible option for systems-level understand- nal datasets under a graph-theoretical framework and ing [1,2,3]. The use of graphs for expression anal- evaluate how cross-stress features can be identified. ysis is inherently an attractive proposition for rea- This is performed both from a node and a network sons related to sparsity, which simplifies the cumber- community organization point of view. Cluster anal- some analysis of large datasets, in addition to being ysis, here viewed as a problem of network partition- mathematically convenient (see examples in Fig.1). ing, is achieved under state-of-the-art algorithms re- The properties of co-expression networks might re- lying on the properties of stochastic processes on the veal a myriad of aspects pertaining to the impact of constructed graphs. -
NGS Raw Data Analysis
NGS raw data analysis Introduc3on to Next-Generaon Sequencing (NGS)-data Analysis Laura Riva & Maa Pelizzola Outline 1. Descrip7on of sequencing plaorms 2. Alignments, QC, file formats, data processing, and tools of general use 3. Prac7cal lesson Illumina Sequencing Single base extension with incorporaon of fluorescently labeled nucleodes Library preparaon Automated cluster genera3on DNA fragmenta3on and adapter ligaon Aachment to the flow cell Cluster generaon by bridge amplifica3on of DNA fragments Sequencing Illumina Output Quality Scores Sequence Files Comparison of some exisng plaGorms 454 Ti RocheT Illumina HiSeqTM ABI 5500 (SOLiD) 2000 Amplificaon Emulsion PCR Bridge PCR Emulsion PCR Sequencing Pyrosequencing Reversible Ligaon-based reac7on terminators sequencing Paired ends/sep Yes/3kb Yes/200 bp Yes/3 kb Read length 400 bp 100 bp 75 bp Advantages Short run 7mes. The most popular Good base call Longer reads plaorm accuracy. Good improve mapping in mulplexing repe7ve regions. capability Ability to detect large structural variaons Disadvantages High reagent cost. Higher error rates in repeat sequences The Illumina HiSeq! Stacey Gabriel Libraries, lanes, and flowcells Flowcell Lanes Illumina Each reaction produces a unique Each NGS machine processes a library of DNA fragments for single flowcell containing several sequencing. independent lanes during a single sequencing run Applications of Next-Generation Sequencing Basic data analysis concepts: • Raw data analysis= image processing and base calling • Understanding Fastq • Understanding Quality scores • Quality control and read cleaning • Alignment to the reference genome • Understanding SAM/BAM formats • Samtools • Bedtools Understanding FASTQ file format FASTQ format is a text-based format for storing a biological sequence and its corresponding quality scores. -
Sequence Data
Sequence Data SISG 2017 Ben Hayes and Hans Daetwyler Using sequence data in genomic selection • Generation of sequence data (Illumina) • Characteristics of sequence data • Quality control of raw sequence • Alignment to reference genomes • Variant Calling Using sequence data in genomic selection and GWAS • Motivation • Genome wide association study – Straight to causative mutation – Mapping recessives • Genomic selection (all hypotheses!) – No longer have to rely on LD, causative mutation actually in data set • Higher accuracy of prediction? • Not true for genotyping-by-sequencing – Better prediction across breeds? • Assumes same QTL segregating in both breeds • No longer have to rely on SNP-QTL associations holding across breeds – Better persistence of accuracy across generations Using sequence data in genomic selection and GWAS • Motivation • Genotype by sequencing – Low cost genotypes? – Need to understand how these are produced, potential errors/challenges in dealing with this data Technology – NextGenSequence • Over the past few years, the “Next Generation” of sequencing technologies has emerged. Roche: GS-FLX Applied Biosystems: SOLiD 4 Illumina: HiSeq3000, MiSeq Illumina, accessed August 2013 Sequencing Workflow • Extract DNA • Prepare libraries – ‘Cutting-up’ DNA into short fragments • Sequencing by synthesis (HiSeq, MiSeq, NextSeq, …) • Base calling from image data -> fastq • Alignment to reference genome -> bam – Or de-novo assembly • Variant calling -> vcf ‘Raw’ sequence to genotypes FASTQ File • Unfiltered • Quality metrics -
BAM Alignment File — Output: Alignment Counts and RPKM Expression Measurements for Each Exon • Calculate Coverage Profiles Across the Genome with “Sam2wig”
01 Agenda Item 02 Agenda Item 03 Agenda Item SOLiD TM Bioinformatics Overview (I) July 2010 1 Secondary and Tertiary Primary Analysis Analysis BioScope in cluster SETS ICS Export BioScope in cloud •Satay Plots •Auto Correlation •Heat maps On Instrument Other mapping tools 2 Auto-Export (cycle-by-cycle) Collect Primary Analysis Generate Secondary Images (colorcalls) Csfasta & qual Analysis Instrument Cluster Auto Export delta spch run definition Merge Generate Tertiary Spch files Csfasta & qual Analysis Bioscope Cluster 3 Manual Export Collect Primary Analysis Generate Secondary Images (colorcalls) Csfasta & qual Analysis Instrument Cluster Option in SETS to export All run data or combination of Spch, csfasta & qual files Tertiary Analysis Bioscope Cluster 4 Auto-Export database requirement removed Instrument Cluster Remote Cluster JMS Broker JMS Broker Export ICS Hades Bioscope (ActiveMQ) (ActiveMQ) Daemon Postgres Postgres SETS Bioscope UI Tomcat Tomcat Disco installer Auto-export installer Bioscope installer System installer 5 SOLiDSOLiD DataData AnalysisAnalysis Workflow:Workflow: Secondary/Tertiary Analysis (off -instrument) Primary Analysis Secondary Analysis Tertiary Analysis BioScope BioScope Reseq WT SAET •SNP •Coverage .csfasta / .qual Accuracy Enhanced .csfasta •InDel •Exon Counting •CNV •Junction Finder •Inversion •Fusion Transcripts Mapping Mapped reads (.ma) Third Party Tools Mapped reads (.bam) maToBam 6 01 Agenda Item 02 Agenda Item 03 Agenda Item SOLiD TM Bioinformatics Overview (II) July 2010 7 OutlineOutline • Color -
Introduction to High-Throughput Sequencing File Formats
Introduction to high-throughput sequencing file formats Daniel Vodák (Bioinformatics Core Facility, The Norwegian Radium Hospital) ( [email protected] ) File formats • Do we need them? Why do we need them? – A standardized file has a clearly defined structure – the nature and the organization of its content are known • Important for automatic processing (especially in case of large files) • Re-usability saves work and time • Why the variability then? – Effective storage of specific information (differences between data- generating instruments, experiment types, stages of data processing and software tools) – Parallel development, competition • Need for (sometimes imperfect) conversions 2 Binary and “flat” file formats • “Flat” (“plain text”, “human readable”) file formats – Possible to process with simple command-line tools (field/column structure design) – Large in size – Space is often saved through the means of archiving (e.g. tar, zip) and human-readable information coding (e.g. flags) • File format specifications (“manuals”) are very important (often indispensable) for correct understanding of given file’s content • Binary file formats – Not human-readable – Require special software for processing (programs intended for plain text processing will not work properly on them, e.g. wc, grep) – (significant) reduction to file size • High-throughput sequencing files – typically GBs in size 3 Comparison of file sizes • Plain text file – example.sam – 2.5 GB (100 %) • Binary file – example.bam – 611 MB (23.36 %) – Possibility of indexing -
Magic-BLAST, an Accurate DNA and RNA-Seq Aligner for Long and Short Reads
bioRxiv preprint doi: https://doi.org/10.1101/390013; this version posted August 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. Magic-BLAST, an accurate DNA and RNA-seq aligner for long and short reads Grzegorz M Boratyn, Jean Thierry-Mieg*, Danielle Thierry-Mieg, Ben Busby and Thomas L Madden* National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, MD, 20894, USA * To whom correspondence should be addressed. Tel: 301-435-5987; Fax: 301-480-4023; Email: [email protected]. Correspondence may also be addressed to [email protected]. ABSTRACT Next-generation sequencing technologies can produce tens of millions of reads, often paired-end, from transcripts or genomes. But few programs can align RNA on the genome and accurately discover introns, especially with long reads. To address these issues, we introduce Magic-BLAST, a new aligner based on ideas from the Magic pipeline. It uses innovative techniques that include the optimization of a spliced alignment score and selective masking during seed selection. We evaluate the performance of Magic-BLAST to accurately map short or long sequences and its ability to discover introns on real RNA-seq data sets from PacBio, Roche and Illumina runs, and on six benchmarks, and compare it to other popular aligners. Additionally, we look at alignments of human idealized RefSeq mRNA sequences perfectly matching the genome. -
Institute for Glycomics Annual Report 2019
INSTITUTE FOR GLYCOMICS ANNUAL REPORT 2019 1 Our MissionFighting diseases of global impact through discovery and translational science. 2 Griffith University | Institute for Glycomics 2019 Annual Report | CRICOS No. 00233E Contents 4 About us 56 2019 publications 6 Director’s report 64 New grants in 2019 8 Institute highlights 67 Remarkable people 9 Remarkable science 68 Membership in 2019 12 Remarkable achievements 71 Plenary/keynote/invited lectures in 2019 18 Translation and commercialisation 73 Remarkable support 20 Commercialisation case study 22 Internationalisation 24 Community engagement 28 Community engagement case studies 30 Selected outstanding publications 40 Research group leader highlights 54 Our facilities Our Vision To be a world-leader in the discovery and development of drugs, vaccines and diagnostics through the application of innovative multidisciplinary science in a unique research environment. 3 About Us The Institute boasts state-of-the-art facilities combined with some of the world’s most outstanding researchers with a significant focus on ‘Glycomics’, a constantly expanding field that explores the structural and functional properties of carbohydrates (sugars). Glycomics research is conducted worldwide in projects that cut across multiple disciplines, applying new approaches to treatment and prevention of diseases. Our unique research expertise makes us the only institute of its kind in Australia and one of very few integrated translational institutes using this approach in the world. The Institute for Glycomics is one of Australia’s flagship multidisciplinary biomedical research institutes. The Institute’s research primarily targets prevention and cures for infectious diseases and cancer, with a focus on translational Established in February 2000, the Institute strives to be research which will inevitably have a positive impact on human a world leader in the discovery and development of next health globally. -
Predicting False Positives of Protein-Protein Interaction Data by Semantic Similarity Measures§ George Montañez1 and Young-Rae Cho*,2
Send Orders of Reprints at [email protected] Current Bioinformatics, 2013, 8, 000-000 1 Predicting False Positives of Protein-Protein Interaction Data by Semantic Similarity Measures§ George Montañez1 and Young-Rae Cho*,2 1Machine Learning Department, Carnegie Melon University, Pittsburgh, PA 15213, USA 2Bioinformatics Program, Department of Computer Science, Baylor University, Waco, TX 76798, USA Abstract: Recent technical advances in identifying protein-protein interactions (PPIs) have generated the genomic-wide interaction data, collectively collectively referred to as the interactome. These interaction data give an insight into the underlying mechanisms of biological processes. However, the PPI data determined by experimental and computational methods include an extremely large number of false positives which are not confirmed to occur in vivo. Filtering PPI data is thus a critical preprocessing step to improve analysis accuracy. Integrating Gene Ontology (GO) data is proposed in this article to assess reliability of the PPIs. We evaluate the performance of various semantic similarity measures in terms of functional consistency. Protein pairs with high semantic similarity are considered highly likely to share common functions, and therefore, are more likely to interact. We also propose a combined method of semantic similarity to apply to predicting false positive PPIs. The experimental results show that the combined hybrid method has better performance than the individual semantic similarity classifiers. The proposed classifier