Answers.Body: <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>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-05-14T19:06:48.340Z
Answers.LastActivityDate: 2019-05-14T19:06:48.340Z
Answers.UserDisplayName: swbarnes2
Body: <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>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-05-14T16:34:00.613Z
Comments.Text: Are you taking transcripts as the reference? `--norc` might be what you are looking for.
Comments.UserDisplayName: user172818
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-05-14T17:12:07.537Z
Comments.Text: I'm using mm10 as my reference (downloaded from Illumina).
Comments.UserDisplayName: irritable_phd_syndrome
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-05-14T18:21:38.617Z
Comments.Text: Then you need an RNA-seq mapper, not bowtie2. STAR and HISAT2 should have options to enable strand specific mapping. I am not familiar with those, though.
Comments.UserDisplayName: user172818
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-05-14T18:38:56.363Z
Comments.Text: Could you please elaborate how bowtie is not an rna-seq mapper? Per their website "Bowtie 2 is often the first step in pipelines for comparative genomics, including for variation calling, ChIP-seq, RNA-seq,"
Comments.UserDisplayName: irritable_phd_syndrome
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-05-14T18:55:16.820Z
Comments.Text: Bowtie2 is not aware of splicing. For typical RNA-seq applications, you will want to use a splice-aware mapper, such as star and hisat2, which is specifically designed for RNA-seq. Tophat2 does use bowtie2 for mapping, but it is invoking bowtie2 in a non-standard way and is generally thought to be superseded by star and hisat2 anyway.
Comments.UserDisplayName: user172818
Tags: <rna-seq><read-mapping><bowtie2>
Title: bowtie2 options when mapping stranded single end reads
ThreadText: # bowtie2 options when mapping stranded single end reads
  
  (Asked by: username_0 on 2019-05-14)
  
  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](http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#default-mode-search-for-multiple-alignments-report-the-best-one) 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?
  
  **Tags:** rna-seq, read-mapping, bowtie2
  
  > **Comments:**
  > **username_1**: Are you taking transcripts as the reference? `--norc` might be what you are looking for.  
  > **username_0**: I'm using mm10 as my reference (downloaded from Illumina).  
  > **username_1**: Then you need an RNA-seq mapper, not bowtie2. STAR and HISAT2 should have options to enable strand specific mapping. I am not familiar with those, though.  
  > **username_0**: Could you please elaborate how bowtie is not an rna-seq mapper? Per their website "Bowtie 2 is often the first step in pipelines for comparative genomics, including for variation calling, ChIP-seq, RNA-seq,"  
  > **username_1**: Bowtie2 is not aware of splicing. For typical RNA-seq applications, you will want to use a splice-aware mapper, such as star and hisat2, which is specifically designed for RNA-seq. Tophat2 does use bowtie2 for mapping, but it is invoking bowtie2 in a non-standard way and is generally thought to be superseded by star and hisat2 anyway.  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_2 on 2019-05-14. Score: 1
  
  I repeat what username_1 says...don't waste your time on bowtie or tophat. Use STAR or HISAT2, or a pseudoaligner like kallisto with a transcriptome.
  
  
  ---
  
  