CuffDiff results does not seem correct
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3.8 years ago

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

RNA-Seq CuffDiff Galaxy • 907 views
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Specific command used and percentage of mapped reads would help.

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@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.

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Are the chromosome identifiers in your reference genome and the gtf the same?

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...is BWA even recommended anywhere for use with Cuffdiff?

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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.

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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.

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