Dear All,
I am trying to analyze my RNAseq data by TopHat-Cufflinks package based on Galaxy. I used Epicentre ScriptSeq strand-specific library construction protocol, which I assume, produces a second-strand library. When I set FR option to "second strand" and run TopHat and Cufflinks it results in confusing transcript orientation. Cufflinks-assembled transcript with multiple exons are oriented as expected. However, transcripts that entirely reside in one exon/intron or intergenic region are labeled in an opposite way. If TopHat accepted hits track contains negative-strand reads (colored red), transcript is labeled as "positive" and vice versa. An example is shown in the attached screenshot. Both transcripts TCONS_00000014 and TCONS_00000015 were mapped to "+" strand, though reads that represent them had been mapped to "-" strand.
Is there a way to solve this issue?
Regards,
Aleks Schein