Aleks,

> I used Epicentre ScriptSeq strand-specific library construction protocol, 
> which I assume, produces a second-strand library.

This an assumption probably worth verifying before going further. The Tophat 
documentation may be helpful as well:

http://tophat.cbcb.umd.edu/manual.html

> 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?

I haven't heard of or encountered this problem before. Your best bet is 
probably to email the Tophat/Cufflinks authors and see if they can shed light 
on your issues:

tophat.cuffli...@gmail.com

Good luck,
J.
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