Cufflinks is a transcript assembly program for RNA-Seq data and it also provides transcript quantification and tools for identifying differentially expressed transcripts.
Cufflinks 1.3.0 is installed at /opt/ and the executable is located at /usr/local/genome/bin/
1. Cufflinks
Basic cufflinks command:
cufflinks accepted_hits.bam -G <gtffile> -o <outputdirectory> &>cufflinks.log &
Input:
- accepted_hits.bam : bam file produced by tophat
- gtffile : gtf file with transcript information
- outputdirectory: Directory to place output files in
Example: For human (hg19)
cufflinks accepted_hits.bam -G /home/daras/data/hg19/refFlat.cufflinks.2.exonsonly.gtf -o geneexp1_output &>cufflinks.log &
/home/daras/data/hg19/refFlat.cufflinks.2.exonsonly.gtf is a human (hg19) refFlat file that has been parsed into a gtf file that is acceptable by cufflinks.'
2. Cuffcompare
Cuffcompare produced a combined gtf file with a nonredundant set of transcripts across multiple samples. This can be used to call differentially expressed transcripts using cuffdiff.
cuffcompare -r <gtffile> <transcripts1.gtf> <transcripts2.gtf>...<transcriptsn.gtf> &>cuffcompare.log &
Input:
- gtffile : gtf file with transcript information that was used in cufflinks
- transcripts1.gtf : cufflinks gtf output for sample1
- transcripts2.gtf : cufflinks gtf output for sample2
3. Cuffdiff
Cuffdiff identifies differentially expressed transcripts.
cuffdiff <transcripts.combined.gtf> <sample1_replicate1.sam[GSAF:,...,sample1_replicateM]> <sample2_replicate1.sam[GSAF:,...,sample2_replicateM.sam]>... [sampleN.sam_replicate1.sam[GSAF:,...,sample2_replicateM.sam]] &>cuffdiff.log &
Example: cuffdiff transcripts.combined.gtf geneexp1_1.sam,geneexp1_2.sam geneexp2_1.sam,geneexp2_2.sam &>cuffdiff.log &
Input:
transcripts.combined.gtf : Combined gtf for all samples output by cuffcompare
sam files : Alignment files for each replicate of sample1 (seperated by commas) followed by Alignment files for each replicate of sample2 (seperated by commas)
