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