text: user2841: 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?
  
  user37: 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.
  
original_text: 2841: <p>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).</p>
  <p>In the bwa output, I see lines <code># candidate unique pairs for (FF, FR, RF, RR): (0, 5372, 2, 0)</code>, which seems to contradict the expected orientation. Is there a reason that this is expected, or does it indicate a problem?</p>
   37: <p>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:</p>
  <pre><code>-------------------&gt;  transcript
    R2 ----&gt;   &lt;---- R1
  </code></pre>
  <p>The RF pair orientation should be</p>
  <pre><code>&lt;----   ----&gt;
  </code></pre>
  <p>You can confirm this by checking the mapping strand of read1.</p>
  