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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