Entering edit mode
6.9 years ago
nazaninhoseinkhan
▴
530
Dear all,
I have RNASeq data obtained from bacterial drug resistant and drug sensitive samples.
I conducted mapping using BWA-MEM in galaxy using the reference genome.
But when I run cuffDiff to obtain the list of differential expressed genes using the gtf file, the value1 and value 2 of all genes are zero and as a result the logFC of all genes are zero too.
Since the mapping step seems to have been performed correctly I suspected to the gtf file that I had used.
The species is Pseudomonas aeruginosa PA14 and I get its genome sequence and gtf file from Pseudomonas genome database.
Can anybody help me to figure out the possible sources of this problem?
Regards
Nazanin
Specific command used and percentage of mapped reads would help.
@OP: Load bam file in IGV and check the reads manually in regions of interest (from GTF) or extract coverage for a region of inteste and check it. I would suggest to check the coverage in house keeping genes.
Are the chromosome identifiers in your reference genome and the gtf the same?
...is BWA even recommended anywhere for use with Cuffdiff?
Not sure how it interacts with Cuffdiff (specific bam tags?) but as an aligner for a prokaryotic organism (without splicing) it would be fine I guess.
Yes I think that the imporant tag would be XS, but not sure. As far as I know, only Bowtie/TopHat adds XS, whereas BWA does not. So, to Cufflinks/CuffDiff, it would be as if there were 0 reads in the BAM.