Answers.Body: <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>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2021-11-23T14:47:59.860Z
Answers.LastActivityDate: 2021-11-23T14:47:59.860Z
Answers.UserDisplayName: user172818
Body: <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>
  
Tags: <rna-seq><phylogenetics><bwa>
Title: Why does BWA MEM orientation contradict my library prep method
ThreadText: # Why does BWA MEM orientation contradict my library prep method
  
  (Asked by: username_0 on 2021-11-21)
  
  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?
  
  **Tags:** rna-seq, phylogenetics, bwa
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2021-11-23. Score: 2
  
  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.
  
  
  ---
  
  