第二代测序中的数据分析-转录组

第二代测序中的数据分析(转录组)6转录组常规分析基因组全基因组/外显子组测序SNPSmallInDelSNPannotation目标区域深度测序SNPannotationDenovo测序Genomeassembly转录组mRNA测序Geneexpression小RNA测序Annotationandtargetprediction6.1常规分析•Transcriptsquantification•Splicingsitesdiscoveryandquantification•Genediscovery•SNP/INDELdetection•Allelespecificexpression•GOandKEGG6.1常规分析Trapnelletal.,20126.1常规分析1.AligntheRNA-seqreadstothegenome–BowtieandTopHat2.Assembleexpressedgenesandtranscripts–Cufflinksandcuffmerge3.Identifydifferentiallyexpressedgenesandtranscripts–Cuffdiff4.Exploredifferentialanalysisresults–CummeRbund6.2分析所需工具•Bowtiesoftware–•SAMtools–•TopHatsoftware–•Cufflinkssoftware–•CummeRbundsoftware–*Linux,64bitCPU,16Gmemory6.3分析准备6.3.1ReadalignmentwithTopHat6.3.1ReadalignmentwithTopHat$Tophat-p8-Ggenes.gtf-oC1_R1_thoutgenomeC1_R1_1.fqC1_R1_2.fq$Tophat-p8-Ggenes.gtf-oC1_R2_thoutgenomeC1_R2_1.fqC1_R2_2.fq$Tophat-p8-Ggenes.gtf-oC1_R3_thoutgenomeC1_R3_1.fqC1_R3_2.fq$Tophat-p8-Ggenes.gtf-oC2_R1_thoutgenomeC2_R1_1.fqC2_R1_2.fq$Tophat-p8-Ggenes.gtf-oC2_R2_thoutgenomeC2_R2_1.fqC2_R2_2.fq$Tophat-p8-Ggenes.gtf-oC2_R3_thoutgenomeC2_R3_1.fqC2_R3_2.fq6.3.2TranscriptassemblywithCufflinks6.3.2TranscriptassemblywithCufflinksStep1:$cufflinks-p8-oC1_R1_cloutC1_R1_thout/accepted_hits.bam$cufflinks-p8-oC1_R2_cloutC1_R2_thout/accepted_hits.bam$cufflinks-p8-oC1_R3_cloutC1_R3_thout/accepted_hits.bam$cufflinks-p8-oC2_R1_cloutC2_R1_thout/accepted_hits.bam$cufflinks-p8-oC2_R2_cloutC2_R2_thout/accepted_hits.bam$cufflinks-p8-oC2_R3_cloutC2_R3_thout/accepted_hits.bamStep2:createafilecalledassemblies.txtthatliststheassemblyfileforeachsample.Step3:$cuffmerge-ggenes.gtf-sgenome.fa-p8assemblies.txt6.3.2TranscriptassemblywithCufflinksCufflinksoutput–FPKMforgene–FPKMfortranscriptisoforms6.3.3DifferentialanalysiswithCuffdiff6.3.3DifferentialanalysiswithCuffdiff$cuffdiff-odiff_out-bgenome.fa-p8-LC1,C2-umerged_asm/merged.gtf./C1_R1_thout/accepted_hits.bam,./C1_R2_thout/accepted_hits.bam,./C1_R3_thout/accepted_hits.bam./C2_R1_thout/accepted_hits.bam,./C2_R3_thout/accepted_hits.bam,./C2_R2_thout/accepted_hits.bam6.3.4VisualizationwithCummeRbundLibrary(cummeRbund)cuff_data-readCufflinks(‘diff_out’)csDensity(genes(cuff_data)csScatter(gene(cuff_data),‘C1’,‘C2’)6.4Downstreamdataanalysis•Functionannotation:GO,Mapman,Interpro,KEGG,Metacyc,etal.•Pathwaymappingandvisulization•Profilingclustering•FunctionenrichmenttestfordifferentialexpressedgenesetDownstreamdataanalysis:GoseqLoughlinetal.7小RNA常规分析基因组全基因组/外显子组测序SNPSmallInDelSNPannotation目标区域深度测序SNPannotationDenovo测序Genomeassembly转录组mRNA测序Geneexpression小RNA测序Annotationandtargetprediction7.1分析常规流程7.2分析所需数据库谢谢!