Transcript RNA-Seq
普通高等教育 “十二五”规划教材 生物信息学 Bioinformatics 第十二章：第二代测序技术 及其应用 Sequence 2.0 Web 2.0 Coined by Darcy DiNucci in 1999. Resurface in 2003. 2004, O'Reilly Media and MediaLive hosted the first Web 2.0 conference. Examples of Web 2.0 include web-based communities, hosted services, web applications, social-networking sites, video-sharing sites, wikis, blogs, mashups and folksonomies. A Web 2.0 site allows its users to interact with other users or to change website content, in contrast to non-interactive websites where users are limited to the passive viewing of information that is provided to them. Ajax/Flash/Flex etc. Next-generation DNA sequencing BAC-based sequencing, whole-genome sequencing (WGS) 1970s-2004, HGP Sanger/Maxam-Gilbert Next-generation DNA sequencing (NGS) 2004-now Second-generation DNA sequencing Deep-sequencing High-throughput DNA sequencing Second-generation DNA sequencing technologies Roche/454 FLX Pyrosequencer Illumina/Solexa Genome Analyzer Applied Biosystems SOLiDTM Sequencer Pacific Sequencer HeliScope Single Molecule Sequencer Roche/454 FLX Pyrosequencer Illumina Genome Analyzer Applied Biosystems SOLiDTM Sequencer Applications of NGS Category Examples of applications Complete genome resequencing Comprehensive polymorphism and mutation discovery in individual human genomes Reduced representation sequencing Large-scale polymorphism discovery Targeted genomic resequencing Targeted polymorphism and mutation discovery Paired end sequencing Discovery of inherited and acquired structural variation Metagenomic sequencing Discovery of infectious and commensal flora RNA-Seq Deep-sequencing shotgun libraries derived from mRNA/small RNAs. microRNA profiling/splice junctions/transcript boundaries/structural rearrangements/copy number variation DNA methylation Determining patterns of cytosine methylation in genomic DNA , large-scale analysis of DNA methylation, by deep sequencing of bisulfite-treated DNA Chromatin immunoprecipitation– sequencing (ChIP-Seq) Genome-wide mapping of protein-DNA interactions, by deep sequencing of DNA fragments pulled down by ChIP. Nuclease fragmentation and sequencing Nucleosome positioning Molecular barcoding Multiplex sequencing of samples from multiple individuals We are interested in … mRNA/small RNA analyses (RNA-Seq) Analysis of mRNA targets of miRNA/siRNA Epigenetic analyses (ChIP-Seq) mRNA targets of miRNA/siRNA Analyses of mRNA targets of miRNA and PARE: mRNA degradation productsparallel by NGS analysis of RNA ends methods. GMUCT: genome-wide mapping of uncapped and leaved transcript Epigenetic analyses based on genome sequence; cross-hybridization (high background levels); limited dynamic range of detection (<1000-fold); normalization problems(across different experiments). Technologies used in transcriptome studies: RNA-Seq Hybridization-based approaches : low throughput; Microarrays/chip; based on expensive Sanger sequencing technology; expensive; b) genomic tiling microarrays. a) high throughput; not quantitative. Sequence-based approaches: more precise; EST: Expression Sequence Tag bp, 20-7000 a a)portion the short tags cannot be(~400 uniquely mapped.bp) b) tag-based methods: CAGE: cap analysis of gene expression (~14-20 bp, 5′ ends) SAGE: serial analysis of gene expression (~14-20 bp, 3′ ends) MPSS: massively parallel signature sequencing (17-20 bp) Next-generation Sequencing-based method: RNA-Seq RNA-Seq Sequencing length: 30 - 400bp. Advantages: can be used to detect transcripts of any genome. low background, highly accurate large dynamic range of expression levels (~10000-fold) high levels of reproducibility (both for technical and biological replicates) requires less RNA sample (cloning steps) lower cost DNA sequencing technologies used for ranscriptome sequencing applications: Illumina IG, Applied Biosystems SOLiD and Roche 454 Life Science systems etc. Advantages of RNA-Seq Advantages of RNA-Seq v.s. other transcriptomics methods RNA-Seq Sequencing length: 30 - 400bp. Advantages: can be used to detect transcripts of any genome. low background, highly accurate large dynamic range of expression levels (~10000-fold) high levels of reproducibility (both for technical and biological replicates) requires less RNA sample (cloning steps) lower cost DNA sequencing technologies used for ranscriptome sequencing applications: Illumina IG, Applied Biosystems SOLiD and Roche 454 Life Science systems etc. RNA-Seq technologies Commercially available sequencing technologies used for transcriptome sequencing applications (Sep 15, 2008). A typical RNA-Seq experiment mapping reads to the genome Program Website Publications BLAST http://www.ncbi.nlm.nih.gov/blast/ 1990, J. Mol. Biol. BLAT http://www.soe.ucsc.edu/~kent/src/ 2002, Genome Research Cross_match http://www.phrap.org/phredphrapconsed.html *** ELAND http://www.illumina.com/ *** TopHat http://tophat.cbcb.umd.edu/ *** Novoalign http://www.novocraft.comne/ *** Mosaik http://bioinformatics.bc.edu/marthlab/Mosaikne/ *** Bowtie http://bowtie.cbcb.umd.edune 2009, Genome Biology BWA http://maq.sourceforge.net/bwa-man.shtmlne/ 2009, Bioinformatics MAQ http://maq.sourceforge.net/ 2008, Genome Research SOAP/SOAP2 http://soap.genomics.org.cn/ 2008/2009, Bioinformatics ZOOM http://www.bioinfor.com/products/zoom/ 2008, Bioinformatics PerM http://code.google.com/p/perm/ 2009, Bioinformatics BWT-SW http://i.cs.hku.hk/~ckwong3/bwtsw/ 2008, Bioinformatics RMAP http://rulai.cshl.edu/rmap/ 2008, BMC Bioinformatics SHRiMP http://compbio.cs.toronto.edu/shrimp/ 2009, PLoS Computational Biology SeqMap http://biogibbs.stanford.edu/~jiangh/SeqMap/ MOM http://mom.csbc.vcu.edu/ ProbMatch http://www.cs.wisc.edu/∼jignesh/probematch/ Exonerate http://www.ebi.ac.uk/~guy/exonerate/ SSAHA2 http://www.sanger.ac.uk/Software/analysis/SSAHA2/ Edena http://www.genomic.ch/edena VCAKE http://sourceforge.net/projects/vcake/ 2007, Bioinformatics Euler-SR *** 2007, Genome Research Mapping algorithms (a) MAQ: based on spaced-seed 2008, Bioinformatics indexing; 2009, Bioinformatics (b) Bowtie: based on 2009, Bioinformatics the Burrows2005, BMC Bioinformatics Wheeler transform 2001, Genome Research (BWT). 2008, Genome Research More Detailed problems Library construction: fragmentation methods. Tag Count 3. mRNAs are reverse transcribed to cDNA, 2. mRNAs randomly fragmented, the fragments 1. mRNAs are randomly fragmented, adapters are PCR amplified, randomly fragmented, andare are5reverse transcribed cDNA, PCR to the and 3 ends,toand then theamplified, adapterthen adapters are attachedattached to the and 3 5 RNA fragmentation andmRNAs then adapters are attached to the 5 and cDNA 3 ends. ligated are reverse transcribed to make ends. and PCR amplified. DNA library preparation: RNA fragmentation and DNA fragmentation compared. Genes More Detailed problems Library construction Gene model coverage by sequencing-based methods for transcriptome analysis and its expression profiling. Reference genome sequence 5’ Extron1 cDNA Intron1 Extron1 Extron2 Extron2 Intron2 3’ Extron3 5’ EST reads of reads No. of reads No. of reads No. ofNo. Extron3 3’ EST CAGE tags Position in the genome SAGE tags RNA-Seq reads Position in the genome Position in the genome Position in the genome More Detailed problems Library construction Gene model coverage by sequencing-based methods for transcriptome analysis and its expression profiling. RNA-Seq used for protein-coding gene annotation. Potential novel intron Reference genome sequence 5’ Extron1 Intron1 Extron2 Sequencing reads Intron2 Mapping Extron3 Potential novel exon More Detailed problems Library construction Gene model coverage by sequencing-based methods for transcriptome analysis and its expression profiling. RNA-Seq used for protein-coding gene annotation. RNA-Seq used for small ncRNAs (miRNA/siRNA) discovery and detection. Transcript rearrangement discovery. Applications of RNA-Seq RNA-Seq can be used to detect the expression profile of small RNAs (miRNA/piRNA/siRNA etc.) and larger RNA (mRNA/tRNA/rRNA etc.). RNA-Seq use for annotation: splice junctions/ transcription (exon/intron) boundaries. RNA-Seq use for mapping of structural rearrangements: translocations, inversions, small insertions/deletions (indels), and copy number variants (CNVs). RNA-Seq can find new genes. RNA-Seq can also reveal sequence variations (SNPs) in the ranscribed regions. Our Works Our Works ChIP-seq ChIP-seq (Chromatin immunoprecipitation followed by sequencing) A technique for genome-wide profiling of DNAbinding proteins, histone modifications or nucleosomes. Illumina Solexa Genome Analyzer (sequencing is performed by sequencing-by-synthesis) Roche 454 platform (sequenced by pyrosequencing) and Applied Biosystems (ABI) SOLiD platform (sequenced DNA ligase-driven synthesis) Helicos HeliScope platform (single-molecule sequencing platform) ChIP-chip ChIP-chip (Chromatin immunoprecipitation followed by hybridization to a microarray) Affymetrix: the ChIP and control samples are hybridized to separate arrays (IP vs. control, twosample and multiple-sample tests) NimbleGen and Agilent: hybridize a ChIP sample and a control sample simultaneously to a single array (log ratios, one-sample test) ChIP-chip and ChIP-seq More detailed prolems Overview of CHiP–seq analysis Software tools for ChIP data analyses Projects ENCODE (ENCyclopedia Of DNA Elements) project: http://www.genome.gov/10005107/ modeNCODE (model organism ENCyclopedia Of DNA Elements) project: http://www.modencode.org/ NIH Roadmap epigenomics Program http://nihroadmap.nih.gov/epigenomics/ GWAS: Genome-Wide Association Studies http://grants.nih.gov/grants/gwas/ The Large-Scale Genome Sequencing Program http://www.genome.gov/10001691/ International HapMap Project http://www.genome.gov/10001688/ Genetic Variation Program http://www.genome.gov/10001551/ 1000 Genomes -- A Deep Catalog of Human Genetic Variation http://www.1000genomes.org/ can highWhat would you do if youWhat could throughput sequencing do for sequence everything? you? Now come to Sequence 2.0 Next-generation Sequencing (NGS). Web 2.0 More than sequence. Platform, resource and analysis. JBrowse: A next-generation genome browser (Dojo library: Ajax) LookSeq: A browser-based web viewer for deep sequencing data (Google Maps: Ajax) References 2008 Medicine 2.0: Social Networking, Collaboration, Participation, Apomediation, and Openness. 2009 Emerging Patient-Driven Health Care Models: An Examination of Health Social Networks, Consumer Personalized Medicine and Quantified Self-Tracking. 2009 RNA-Seq: a revolutionary tool for transcriptomics. 2009 Applications of New Sequencing Technologies for Transcriptome Analysis. 2008 Next-Generation DNA Sequencing Methods. 2008 Next-generation DNA sequencing. 2005 Next generation sequencing technologies. 2008 Applications of next-generation sequencing technologies in functional genomics. 2008 What would you do if you could sequence everything? 2006 Genome-Wide Analysis of Protein-DNA Interactions. 2008 Design and analysis of ChIP-seq experiments for DNA-binding proteins. 2009 Insights from genomic profiling of transcription factors. 2008 An integrated software system for analyzing ChIP-chip and ChIP-seq data. 2009 ChIP-seq: Using high-throughput sequencing to discover protein–DNA interactions. 2009 Can web 2.0 reboot clinical trials. 2009 LookSeq: A browser-based viewer for deep sequencing data. 2009 JBrowse: A next-generation genome browser. 2009 PMRD: plant microRNA database. 2009 TransmiR: a transcription factor–microRNA regulation database