Cufflinks problem

After running tophat, we get accepted_hits.bam file

1. /home/xusheng/Biosoft/cufflinks-1.0.3.Linux_x86_64/cufflinks -p 16 -g /home/xusheng/GenomeSequence/Human/Homo_sapiens.GRCh37.63.gtf Nor3_accepted_hits.bam

/home/xusheng/Biosoft/cufflinks-1.0.3.Linux_x86_64/cufflinks -p 16 -g Genes_July_2010_hg19.gff AD3_accepted_hits.bam (gff file only contain gene info)

remember: 1. use the UCSC gff file or ENSEMBL gff file; 2. specify the sequence file when performing the cuffcompare analysis, as the following command; 3. use -g rather than -G


3. cuffcompare step
/home/xusheng/Biosoft/cufflinks-1.0.3.Linux_x86_64/cuffcompare -o adnor_compare -s /home/xusheng/GenomeSequence/Human/Ensembl/Hs19_Ensembl.fa -r /home/xusheng/GenomeSequence/Human/Homo_sapiens.GRCh37.63.gtf AD1/AD1_transcripts.gtf AD3/AD3_transcripts.gtf AD4/AD4_transcripts.gtf AD6/AD6_transcripts.gtf AD7/AD7_transcripts.gtf Nor3/Nor3_transcripts.gtf Nor4/Nor4_transcripts.gtf Nor5/Nor5_transcripts.gtf Nor7/Nor7_transcripts.gtf

good links:

http://dingo.ucsf.edu/twiki/bin/view/Cores/BioinformaticsCore/RnaSeqAnalysis


tophat-cufflinks-cuffcompare-cuffdiff (2011-03-29 15:28:55)转载
标签： 杂谈 分类： Nextgenerationsequencing
1. Creation of a genome index (needed for Bowtie and TopHat to operate)
bowtie-build genome.fa bowtie_output/genome
2. Alignments of RNA-seq reads to the genome with identification of exon-exon splice functions using TopHat
tophat -o tophat_output/ bowtie_output/genome reads.fastq
3. Assembly of RNA-seq reads into transcripts and report of the RPKM/FPKM using Cufflinks
cufflinks -o cufflinks_output/ --reference-seq genome.fa tophat_output/accepted_hits.bam
4. Comparison of assembled transcripts with a reference annotation, or across experiments, using Cuffcompare
cuffcompare -o cuffcompare_output -r genome.gtf -R cufflinks_output/transcripts.gtf
5. Finding significant changes in transcript expression, splicing, and promoter use using Cuffdiff
cuffdiff -o cuffdiff_output/ cufflinks_output/transcripts.gtf tophat_output/accepted_hits1a.bam,tophat_output/accepted_hits1b.bam tophat_output/accepted_hits2.bam
(in this example, accepted_hits1a.bam and accepted_hits1b.sam are replicates, while accepted_hits2.bam is another experimental condition)