第二代测序中的数据分析-转录组

6.3.2 Transcript assembly with Cufflinks
6.3.2 Transcript assembly with Cufflinks
Step 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
7.1 分析常规流程
7.2 分析所需数据库
谢谢 !
ຫໍສະໝຸດ Baidu
6.1 常规分析
• Transcripts quantification • Splicing sites discovery and quantification • Gene discovery • SNP/INDEL detection • Allele specific expression • GO and KEGG
$ 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
6.4 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
• SAM tools
–
• TopHat software
–
• Cufflinks software
–
• CummeRbund software
–
*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
http://bowtie-bio.sourceforge.net/index.shtml/ http://samtools.sourceforge.net/ http://tophat.cbcb.umd.edu/ http://cufflinks.cbcb.umd.edu/ http://compbio.mit.edu/cummeRbund/
Downstream data analysis: Goseq
Loughlin et al.
7 小 RNA 常规分析
SNP 全基因组 / 外显子组测序 基因组 目标区域深度测序 De novo 测序 mRNA 测序 转录组 小 RNA 测序 Small InDel SNP annotation SNP annotation Genome assembly Gene expression Annotation and target prediction
Step2: create a file called assemblies.txt that lists the assembly file for each sample.
Step3: $ cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt
$ 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
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
–
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
第二代测序中的数据分析 ( 转录组 )
罗奇斌
6 转录组常规分析
SNP 全基因组 / 外显子组测序 基因组 目标区域深度测序 De novo 测序 mRNA 测序 转录组 小 RNA 测序 Small InDel SNP annotation SNP annotation Genome assembly Gene expression Annotation and target prediction
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
6.3.4 Visualization with CummeRbund
> > > > Library (cummeRbund) cuff_data <- readCufflinks(‘diff_out’) csDensity(genes(cuff_data) csScatter(gene(cuff_data), ‘C1’, ‘C2’)