Cufflinks

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)