text: user7167: I've got some doubts on the hisat2 --rna-strandness option and its output for downstream analysis. Is it expected to see a difference in alignment and counting results given the default usage of --rna-strandness in hisat2 followed by htseq-count -s reverse on a strand-specific assay?
  Please see below.
  I understand that the --rna-strandness option produces an XS tag to indicate where a transcript is from (on the + or - strand) for downstream transcriptome assembly analysis. I have a paired-end stranded sequencing library that was aligned to the genome using hisat2 without specifying the --rna-strandness (in other words, the default unstranded was the usage). Following this, the reads were assigned to genes using htseq-count and this time -s reverse was specified given the strand-specific sequencing assay type.
  Since --rna-strandness is for transcriptome assembly using the XS tags generated and htseq does not use XS tags for counting, I presume there should be no practical impact from the above. Could you also shed light on this? in case I may have been overlooking other facts of the usages of the tools.
  To help verify the above, I re-aligned and counted the reads from 2 samples by switching on --rna-strandness RF in hisat2. I attach the alignment and count features info. below for assessment.
  Overall alignment rate of Sample 1:
  94.52% (--rna-strandness RF)
  94.12% (--rna-strandness unstranded)
  
  Overall alignment rate of Sample 2:
  94.57% (--rna-strandness RF)
  94.15% (--rna-strandness unstranded)
  
  Feature counts of Sample 1 (following --rna-strandness RF + -s reverse):
  __no_feature        6327294
  __ambiguous     2954776
  __too_low_aQual     3784481
  __not_aligned       688856
  __alignment_not_unique      4858182
  
  Feature counts of Sample 1 (following --rna-strandness unstranded + -s reverse):
  __no_feature        6291151
  __ambiguous     2911298
  __too_low_aQual     4075017
  __not_aligned       754400
  __alignment_not_unique      16136045
  
  Feature counts of Sample 2 (following --rna-strandness RF + -s reverse):
  __no_feature        5417882
  __ambiguous     1708510
  __too_low_aQual     3532352
  __not_aligned       564596
  __alignment_not_unique      2859501
  
  Feature counts of Sample 2 (following --rna-strandness unstranded + -s reverse):
  __no_feature        5359434
  __ambiguous     1676091
  __too_low_aQual     3813344
  __not_aligned       623122
  __alignment_not_unique      2891792
  
  These results look comparable to me across pipelines.
  Thanks
  Guan
  
  user2388: If you reran the command with the correct settings, just leave it at that.  (It is not at all clear to me that strandedness rf is correct) 
  If you want people to tell you if you ran the commands right, you need to put down what commands you used.
  
  user383: Sample 2 looks comparable but Sample 1 shows a drastic reduction in unique alignments.
  Your strandedness param is going to dictate how your alignments are done in the sense that discordant pairs will either be detected or not.
  I would identify the strandedness of your library and use the appropriate respective param.
  
original_text: 7167: <p>I've got some doubts on the hisat2 <code>--rna-strandness</code> option and its output for downstream analysis. Is it expected to see a difference in alignment and counting results given the default usage of <code>--rna-strandness</code> in hisat2 followed by <code>htseq-count -s reverse</code> on a strand-specific assay?</p>
  <p>Please see below.</p>
  <p>I understand that the <code>--rna-strandness</code> option produces an XS tag to indicate where a transcript is from (on the + or - strand) for downstream transcriptome assembly analysis. I have a paired-end stranded sequencing library that was aligned to the genome using hisat2 without specifying the <code>--rna-strandness</code> (in other words, the default unstranded was the usage). Following this, the reads were assigned to genes using htseq-count and this time <code>-s reverse</code> was specified given the strand-specific sequencing assay type.</p>
  <p>Since <code>--rna-strandness</code> is for transcriptome assembly using the XS tags generated and htseq does not use XS tags for counting, I presume there should be no practical impact from the above. Could you also shed light on this? in case I may have been overlooking other facts of the usages of the tools.</p>
  <p>To help verify the above, I re-aligned and counted the reads from 2 samples by switching on <code>--rna-strandness RF</code> in hisat2. I attach the alignment and count features info. below for assessment.</p>
  <p>Overall alignment rate of Sample 1:</p>
  <pre><code>94.52% (--rna-strandness RF)
  94.12% (--rna-strandness unstranded)
  </code></pre>
  <p>Overall alignment rate of Sample 2:</p>
  <pre><code>94.57% (--rna-strandness RF)
  94.15% (--rna-strandness unstranded)
  </code></pre>
  <p>Feature counts of Sample 1 (following --rna-strandness RF + -s reverse):</p>
  <pre><code>__no_feature        6327294
  __ambiguous     2954776
  __too_low_aQual     3784481
  __not_aligned       688856
  __alignment_not_unique      4858182
  </code></pre>
  <p>Feature counts of Sample 1 (following --rna-strandness unstranded + -s reverse):</p>
  <pre><code>__no_feature        6291151
  __ambiguous     2911298
  __too_low_aQual     4075017
  __not_aligned       754400
  __alignment_not_unique      16136045
  </code></pre>
  <p>Feature counts of Sample 2 (following --rna-strandness RF + -s reverse):</p>
  <pre><code>__no_feature        5417882
  __ambiguous     1708510
  __too_low_aQual     3532352
  __not_aligned       564596
  __alignment_not_unique      2859501
  </code></pre>
  <p>Feature counts of Sample 2 (following --rna-strandness unstranded + -s reverse):</p>
  <pre><code>__no_feature        5359434
  __ambiguous     1676091
  __too_low_aQual     3813344
  __not_aligned       623122
  __alignment_not_unique      2891792
  </code></pre>
  <p>These results look comparable to me across pipelines.</p>
  <p>Thanks
  Guan</p>
   2388: <p>If you reran the command with the correct settings, just leave it at that.  (It is not at all clear to me that strandedness rf is correct) </p>
  
  <p>If you want people to tell you if you ran the commands right, you need to put down what commands you used.</p>
   383: <p>Sample 2 looks comparable but Sample 1 shows a drastic reduction in unique alignments.</p>
  <p>Your strandedness param is going to dictate how your alignments are done in the sense that discordant pairs will either be detected or not.</p>
  <p>I would identify the strandedness of your library and use the appropriate respective param.</p>
  