<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>

CC BY-SA 4.0
2021-11-23T14:47:59.860Z
2021-11-23T14:47:59.860Z
user172818
<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>

<rna-seq><phylogenetics><bwa>
Why does BWA MEM orientation contradict my library prep method
# 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

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## 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.


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