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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--
Jennifer Jackson
http://usegalaxy.org/
http://galaxyproject.org/
___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org. Please keep all replies on the list by
using "reply all" in your mail client. For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists,
please use the interface at:

http://lists.bx.psu.edu/