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
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
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