Hello David,

There is most likely some mismatch between the input data. Some things to check:


First, double check that the identifiers in the reference genome exactly match those in the RefGene gtf file and modify if necessary.

Second, make certain that your RefGene file is sorted the same as the sorting in the BAM file (usually by reference genome chromosome/scaffold/etc and start coordinate).

If neither of these solve the issue, Cufflinks has an email list for questions: tophat.cufflinks at gmail.com

These are the essentials from this prior Q/A thread concerning the same issue:
http://lists.bx.psu.edu/pipermail/galaxy-user/2011-March/002267.html

Hopefully this helps! Best wishes for your project,

Jen
Galaxy team


On 7/19/11 12:52 PM, David K Crossman wrote:
Hello!

I have come across a problem where Cufflinks is reporting all FPKM
values as zeroes (0). I have a unique RNA-Seq project from a
collaborator where they are studying eyesight by using tree shrews. I
found that Ensembl
(http://useast.ensembl.org/Tupaia_belangeri/Info/Index) has the FASTA
file for the tree shrew genome (only a 2x coverage, so not very good in
the first place) and had this file indexed in our local instance of
Galaxy. I ran TopHat and it looks as if TopHat ran fine because I’m
getting anywhere from 71-80% properly paired when I check the stats
using “Flagstat.” I then take the accepted hits BAM file from TopHat
plus the GTF RefGene file from Ensembl for tree shrew and load that into
Cufflinks. It seems as if Cufflinks works okay, but when I inspect
Cufflinks three output files, all the FPKM values are 0.

I have two other RNA-Seq projects (human and mouse) and both of these
projects worked fine through TopHat and Cuff(links/Compare/Diff) and
with a RefGene GTF file on our local instance of Galaxy (as well as on
the Galaxy instance at Penn State), so it makes me think that both
TopHat and Cufflinks are working okay.

I’m wondering if it has to do something with the tree shrew reference
genome. Has anyone encountered anything like this? If so, how did you
solve the problem? If not, do you have any suggestions as to what I can
do next? Any help/info would be greatly appreciated.

Thanks,

David



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