text: user3886: I'm trying to map RNA-Seq reads generated using the NEB Ultra Directional kit is first strand reversed protocal. I'm using bowtie-2.3.5_1 to align against the mus musculuous (mm10) genome.  The reads are single ended.
  It seems that --fr and --rf are relevant only for paired end options. I tried these options as well as not specifying either (default).
  E.g.
  $ bowtie2 --fr \
          --threads 20 \
          --very-sensitive \
          -x /reference/mus_musculus/mm10/ucsc_assmebly/illumina_download/Sequence/Bowtie2Index/genome \
          -U trimmed_out/my_trimmed.fq.gz \
          -S bowtie_fr_1.sam
  
  $ samtools sort -n -o bowtie_fr_sort_1.sam bowtie_def_1.sam
  
  I then compared the output of bowtie_rf_sort.sam bowtie_default_sort.sam and bowtie_fr_sort.sam.  They are basically identical excluding the header.
  Question:
  Does strandedness matter in this alignment? Perhaps I should care about the --nofw/--norc options? Am I missing something here?
  
  user2388: I repeat what user172818 says...don't waste your time on bowtie or tophat.  Use STAR or HISAT2, or a pseudoaligner like kallisto with a transcriptome.
  
original_text: 3886: <p>I'm trying to map RNA-Seq reads generated using the <code>NEB Ultra Directional kit is first strand reversed</code> protocal. I'm using <a href="http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#default-mode-search-for-multiple-alignments-report-the-best-one" rel="nofollow noreferrer">bowtie-2.3.5_1</a> to align against the mus musculuous (mm10) genome.  The reads are single ended.</p>
  
  <p>It seems that <code>--fr</code> and <code>--rf</code> are relevant only for paired end options. I tried these options as well as not specifying either (default).</p>
  
  <p>E.g.</p>
  
  <pre><code>$ bowtie2 --fr \
          --threads 20 \
          --very-sensitive \
          -x /reference/mus_musculus/mm10/ucsc_assmebly/illumina_download/Sequence/Bowtie2Index/genome \
          -U trimmed_out/my_trimmed.fq.gz \
          -S bowtie_fr_1.sam
  
  $ samtools sort -n -o bowtie_fr_sort_1.sam bowtie_def_1.sam
  </code></pre>
  
  <p>I then compared the output of <code>bowtie_rf_sort.sam</code> <code>bowtie_default_sort.sam</code> and <code>bowtie_fr_sort.sam</code>.  They are basically identical excluding the header.</p>
  
  <p><strong>Question</strong>:</p>
  
  <p>Does strandedness matter in this alignment? Perhaps I should care about the <code>--nofw/--norc</code> options? Am I missing something here?</p>
   2388: <p>I repeat what user172818 says...don't waste your time on bowtie or tophat.  Use STAR or HISAT2, or a pseudoaligner like kallisto with a transcriptome.</p>
  