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 SNP Small InDel SNP annotation 目标区域深度测序SNP annotation De novo 测序Genome assembly 转录组 mRNA 测序Gene expression 小 RNA 测序 Annotation and target prediction 6.1 常规分析 Transcripts quantification Splicing sites discovery and quantification Gene discovery SNP/INDEL detection Allele specific expression GO and KEGG 6.1 常规分析 Trapnell et al., 2012 6.1 常规分析 1. Align the RNA-seq reads to the genome Bowtie and TopHat 2. Assemble expressed genes and transcripts Cufflinks and cuffmerge 3. Identify differentially expressed genes and transcript s Cuffdiff 4. Explore differential analysis results CummeRbund 6.2 分析所需工具 Bowtie software http:/bowtie- SAM tools TopHat software / Cufflinks software / CummeRbund software /cummeRbund/ *Linux, 64bit CPU, 16G memory 6.3 分析准备 6.3.1 Read alignment with TopHat 6.3.1 Read alignment with TopHat $ Tophat -p 8 -G genes.gtf -o C1_R1_thout genome C1_R1_1.fq C1_R1_2.fq $ Tophat -p 8 -G genes.gtf -o C1_R2_thout genome C1_R2_1.fq C1_R2_2.fq $ Tophat -p 8 -G genes.gtf -o C1_R3_thout genome C1_R3_1.fq C1_R3_2.fq $ Tophat -p 8 -G genes.gtf -o C2_R1_thout genome C2_R1_1.fq C2_R1_2.fq $ Tophat -p 8 -G genes.gtf -o C2_R2_thout genome C2_R2_1.fq C2_R2_2.fq $ Tophat -p 8 -G genes.gtf -o C2_R3_thout genome C2_R3_1.fq C2_R3_2.fq 6.3.2 Transcript assembly with Cufflinks 6.3.2 Transcript assembly with Cufflinks Step 1Step 1: $ cufflinks -p 8 -o C1_R1_clout C1_R1_thout/accepted_hits.bam $ cufflinks -p 8 -o C1_R2_clout C1_R2_thout/accepted_hits.bam $ cufflinks -p 8 -o C1_R3_clout C1_R3_thout/accepted_hits.bam $ cufflinks -p 8 -o C2_R1_clout C2_R1_thout/accepted_hits.bam $ cufflinks -p 8 -o C2_R2_clout C2_R2_thout/accepted_hits.bam $ cufflinks -p 8 -o C2_R3_clout C2_R3_thout/accepted_hits.bam Step2Step2: create a file called assemblies.txt that lists the assembly file for each sample. Step3Step3: $ cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt 6.3.2 Transcript assembly with Cufflinks Cufflinks output FPKM for gene FPKM for transcript isoforms 6.3.3 Differential analysis with Cuffdiff 6.3.3 Differential analysis with Cuffdiff $ cuffdiff -o diff_out -b genome.fa -p 8 -L C1, C2 -u merged_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.bam 6.3.4 Visualization with CummeRbund Library (cummeRbund) cuff_data csDensity(genes(cuff_data) csScatter(gene(cuff_data), C1, C2) 6.4 Downstream data analysis Function annotation: GO, Mapman, Interpro, KEGG, Metacyc, et al. Pathway mapping and visulization Profiling clustering Function enrichment test for differential expressed gene set Downstream data analysis: Goseq Loughlin et al. 7 小 RNA 常规分析 基因组 全基因组 / 外显子组测序 SNP Small InDel SNP anno