Cuffcompare to add tss_id to GTF

[WHP]

Hi all,

I am currently trying to run cuffdiff for a DE analysis on RNA seq stranded reads aligned to transcriptome using bowtie2. I first downloaded a gtf file from Flybase and ran the program with 6 samples (2 genotypes in triplicate) and it gave me very odd results. There was no DE expressed genes and the abundances for a transcript were either 0 or a ridculously high number, and the same number for each transcript. So, I figured it might be the gtf file.

On the cufflinks website it says to run the following if you use a different gtf file than cufflinks produced:

" cuffcompare -s /path/to/genome_seqs.fa -CG -r annotation.gtf annotation.gtf "

So, I took a fasta file for each chromosomal sequence and the original gtf file that didn't work, and ran that command. What I am wondering (as it runs, not sure if it will work yet) is what EXACTLY that command actually does? Does it only add the tss_id to the original gtf file? Or does it reorganize it in any other way? Why do I have to put the "annotation.gtf" file twice in this command?

Thanks,

Billy

[WHP]

Also, any confirmation that I've done the correct thing would be well recieved as well