<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>

CC BY-SA 4.0
2019-05-14T19:06:48.340Z
2019-05-14T19:06:48.340Z
swbarnes2
<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>

CC BY-SA 4.0
2019-05-14T16:34:00.613Z
Are you taking transcripts as the reference? `--norc` might be what you are looking for.
user172818
CC BY-SA 4.0
2019-05-14T17:12:07.537Z
I'm using mm10 as my reference (downloaded from Illumina).
irritable_phd_syndrome
CC BY-SA 4.0
2019-05-14T18:21:38.617Z
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.
user172818
CC BY-SA 4.0
2019-05-14T18:38:56.363Z
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,"
irritable_phd_syndrome
CC BY-SA 4.0
2019-05-14T18:55:16.820Z
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.
user172818
<rna-seq><read-mapping><bowtie2>
bowtie2 options when mapping stranded single end reads
# 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 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.  

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## 1 answers

### Answer by username_2 on 2019-05-14. Score: 1

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.


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