12国外大学研究所收集谷歌ngs pipeline的.pptxVIP

RNAseq Applications in Genome StudiesAlexander Kanapin, PhDWellcome Trust Centre for Human Genetics,University of OxfordRNAseq ProtocolsNext generation sequencing protocol cDNA, not RNA sequencingTypes of libraries available:Total RNA sequencingpolyA+ RNA sequencingSmall RNA sequencingSpecial protocols:DSN treatmentRibosomal depletionGenome Study Applicationstranscriptome analysisidentifying new transcribed regions expression profilingResequencing to find genetic polymorphisms: SNPs, micro-indels CNVscDNA SynthesisSequencing detailsStandard sequencingpolyA/total RNASize slectionPrimers and adaptersSingle- and paired-end sequencingStrand-specific sequencingBeta versionSequencing only + or – strandMostly paired-endArrays vs RNAseq (1)Correlation of fold change between arrays and RNAseq is similar to correlation between array platforms (0.73)Technical replicates are almost identical, no need to runExtra analysis: prediction of alternative splicing, SNPsLow- and high-expressed genes do not match Array vs RNAseq (2)A bit of statisticsShort reads distributionPoisson Negative binomialNormalExpression values normalizationFPKMNormalized reads numberVST (variance stabilized transformation)Differential expression analysisReplicates vs non-replicatesAnalysis DataflowIllumina Pipeline(FASTQ)Alignment (BAM)FASTX Toolkit(FASTQ/FASTA)Expression profiles/RNA abundanceSNP analysisSplice variantsSoftwareShort reads alignersStampy, BWA, Novoalign, Bowtie,…Data preprocessing (reads statistics, adapter clipping, formats conversion, read counters)Fastx toolkitHtseqMISOsamtoolsExpression studiesCufflinks packageRSEQtoolsR packages (DESeq, edgeR, baySeq, DEGseq, Genominator)Alternative splicingCufflinksAugustusCommercial softwarePartekCLCBioFASTQ: Sequence Data“FASTA with Qualities” @HWI-EAS225:3:1:2:854#0/1 GGGGGGAAGTCGGCAAAATAGATCCGTAACTTCGGG +HWI-EAS225:3:1:2:854#0/1 a`abbbbabaabbababb^`[aaa`_N]b^ab^``a @HWI-EAS225:3:1:2:1595#0/1 GGGAAGATCTCAAAAACAGAAGTAAAACATCGAACG +HWI-EAS225:3:1:2:159