Mixing paired-end and single-end reads in Tophat: Do I have to reverse the SE-reads f

[JonB]

This is a cross-post from the Tuxedo Google group: https://groups.google.com/forum/#!to...rs/BtI3_fp_n-o

And a follow-up from a previous post


Hope my question makes sense. I have paired-end reads, but after trimming some reads are left as single-end. I know that tophat can take a mix of paired-end and single-end reads, but do I have to reverse the single-end reads from the rightmost pair (usually called the R2-file or something similar) if they have no mate?

Thanks,

Jon

[SylvainL]

Not sure I understood, but all the reads are from 5' to 3' so you should not have to reverse the SE...

[JonB]

The paired reads are sequenced like this
R1------> <------R2
and I guess tophat takes this into account when it is fed paired reads. But when it only gets the R2 reads as single end, without knowing that they were part of a pair, will it still be ok?

[SylvainL]

If your R1 read is mapping to the forward strand, then the R2 will map to the reverse one...

Actually, the only interest to reverseComplement (not only reverse) would be if you did stranded RNAseq... but even in this case, if you map separately only the R2 reads, you can simply change the strand afterwards...

[JonB]

I should have mentioned that I do have stranded sequencing. So I guess I will map the R2 reads separately.
Thanks!

[SylvainL]

Ok, then it makes sense to reverseComplement (or easiest, as mentioned, is to change the strand after the mapping for R2 alone...)...
Actually, maybe to confuse you a bit more, the R2 (without reverseComplementing them, or witghout changing the strand after mapping), will be on the same strand than your gene (the coding sequence). R1 will be in the opposite one