I have some RNA-seq data from a stranded paired end library prep, with dUTP and UDG preparation, so the orientation should be RF (confirmed with sequencing provider). I assembled the reads with Trinity, specifying RF for the strandedness, and I am mapping the reads to the transcripts with bwa-mem (I am aware bowtie2 or STAR might be better for this, but bwa is what I presently have installed, so just using it to check).

In the bwa output, I see lines # candidate unique pairs for (FF, FR, RF, RR): (0, 5372, 2, 0), which seems to contradict the expected orientation. Is there a reason that this is expected, or does it indicate a problem?


1 Answer 1


Not familiar with the protocol, but I guess RF means different things here. If I am right, with your RF protocol, the first read comes from the reverse strand of the transcript and the second read from the forward strand, but the pair orientation is still FR:

------------------->  transcript
  R2 ---->   <---- R1

The RF pair orientation should be

<----   ---->

You can confirm this by checking the mapping strand of read1.


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