We have discovered a couple of examples where variants were predicted to affect normal splicing but Tophat2/Bowtie failed to identify the effect on splicing within the RNA-Seq alignments.
Specifically we have two recent examples, one of which resulted in a 2bp loss at the 5 prime end of a known exon, and another where the 5 prime end of the known exon was extended by several bases through intron retention. In neither case was the variant present in the spliced transcript therefore the variants themselves should not confuse the alignment.
In each case, the aligner produced no alignments for the affected reads (One member of the read pair is aligned but the one covering the affected splice junction is not aligned at all. Choosing to BLAT the unaligned read in IGV reveals the new splice junction as expected)
We have successfully detected many similar examples in the past and are not sure what may be different about these cases. We subsequently ran STAR aligner and it detected the aberrant splicing without issue.
I am wondering what settings might be adjustable in order to enable Bowtie/Tophat2 to correctly identify the aberrant splicing.
Does anyone have suggestions of what parameters we might look at first? More details available on request,
You probably know this already, but tophat2 has been superseded with hisat2 (or STAR if you prefer). Did you try these more recent tools?
We tried STAR aligner and it detected both events. Problem is that Bowtie is ingrained in some of our pipelines so I was hoping to figure out whether it could be parameterized to make the calls.