Using Cuffdiff With Merged Transcripts (Cuffmerge-Merge.Gtf)

Question

[Sahel]

Hi all,

I have 6 tumor and 6 normal RNA-seqs. and I would like to use cuffdiff to find differentially expressed transcripts (specially novel transcripts).

So far, I used cufflink and then cuffmerge to merge transcript.cfg files for each condition. so I have two merge.gtf files, one for tumor and one for normal samples.

My question is how to use merge.gtf files in my command for cuffdiff?

I use the following command, but it does not work:

cuffdiff -p 4 -o FV_FP_cuffdiff -L FV,FP ~/hg19_index/hg19.gtf -u ~/merge_FV/cuffmerge_FVout/merged.gtf,~/merge_FP/cuffmerge_FPout/merged.gtf ~/tophat_out1/accepted_hits.bam,~/tophat_out2/accepted_hits.bam,~/tophat_out3/accepted_hits.bam,~/tophat_out4/accepted_hits.bam,~/tophat_out5/accepted_hits.bam,~/tophat_out6/accepted_hits.bam ~/tophat_out7/accepted_hits.bam,~/tophat_out8/accepted_hits.bam,~/tophat_out9/accepted_hits.bam,~/tophat_out10/accepted_hits.bam,~/tophat_out11/accepted_hits.bam,~/tophat_out12/accepted_hits.bam

Using merge files has been mentioned in the cufflink manual (The main purpose of this (cuffmerge) script is to make it easier to make an assembly GTF file suitable for use with Cuffdiff) and this Rna-Seq With Cuffdiff: Use Merged.Gtf From Cuffmerge Or Combined.Gtf From Cuffcompare?, but I could not find a practical example.

Thanks very much in advance,

[Michael]

Undeleted, please don't delete questions which have answers.

Answer 1

[Graslevy]

As you can see from the usage example on Cufflinks page,: cuffdiff [options]* transcripts.gtf sample1_replicate1.sam[,...,sample1_replicateM]> <sample2_replicate1.sam[,...,sample2_replicatem.sam]... [samplen.sam_replicate1.sam[,...,sample2_replicatem.sam]]<="" p="">

your merge.gtf file will replace transcripts.gtf. You may also need to convert to SAM output or request SAM during tophat run.