<p>You should be using Bowtie 2 instead of bowtie:</p>
<blockquote>
<p>Bowtie 1 was released in 2009 and was geared toward aligning the relatively short sequencing reads (up to 25-50 nucleotides) prevalent at the time. Since then, technology has improved both sequencing throughput (more nucleotides produced per sequencer per day) and read length (more nucleotides per read).</p>
</blockquote>
<p><a href="http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#how-is-bowtie-2-different-from-bowtie-1" rel="nofollow noreferrer">http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#how-is-bowtie-2-different-from-bowtie-1</a></p>
<p>In particular, Bowtie 2 supports local alignment, which is <em>essential</em> when mapping microRNA. All microRNA sequences are short, which means that there will be adapter read-through in the sequenced reads. This adapter sequence will not map to the genome, and that will cause global aligners (like Bowtie) to fail.</p>
<p>An alternative approach would be to trim off <a href="http://www.usadellab.org/cms/?page=trimmomatic" rel="nofollow noreferrer">palindromic adapter sequences</a> prior to aligning (which it is possible you've tried to do given the file name). Because of adapter read through, standard adapter trimming will not be sufficient. What needs to be trimmed off is the <em>reverse-complement</em> of the adapter sequences. I have no experience using <em>fastp</em>, so don't know if it is capable of doing that.</p>
<p>However, even after trimming there will still be some additional short sequences left on some reads, so a local alignment would still be a good idea.</p>
<p>Doing a local alignment with Bowtie 2 is much easier and faster than trying to troubleshoot adapter read-through issues. The command line you would run is similar, but not identical:</p>
<pre><code>bowtie2-build -f miRNA.fa miRNA
bowtie2 --local -x miRNA -1 SRR16643593_1.NEW.trim.fq -2 SRR16643593_2.NEW.trim.fq | samtools sort &gt; test.bam
</code></pre>

CC BY-SA 4.0
2021-11-30T21:37:08.137Z
2021-11-30T21:43:18.167Z
2021-11-30T21:43:18.167Z
gringer
<p><em>This question was also asked on <a href="https://www.biostars.org/p/9499558/" rel="nofollow noreferrer">Biostars</a></em></p>
<p>I am working on miRNA alignment using bowtie. But it seems to fail to map. Here are the commands:</p>
<pre><code>bowtie-build -f miRNA.fa miRNA
bowtie --norc -k 1 -v 0 /public/home/wschen_gibh/cpa-seq/ref/miRNA/miRNA -1 SRR16643593_1.NEW.trim.fq -2 SRR16643593_2.NEW.trim.fq -S test.sam 
</code></pre>
<p>and here is the result:
<a href="https://i.sstatic.net/FpPCI.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/FpPCI.png" alt="enter image description here" /></a></p>
<p>and the output sam file:
<a href="https://i.sstatic.net/bFd3I.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/bFd3I.png" alt="enter image description here" /></a></p>

CC BY-SA 4.0
2021-11-30T07:12:34.773Z
Ah, this is "CAP-seq", so there are presumably UMIs in those sequences that you need to deal with first
Devon Ryan
CC BY-SA 4.0
2021-11-30T18:37:51.387Z
it would help to see the reference file. Can you confirm that you actually expect anything to align to that reference?
Maximilian Press
<phylogenetics>
bowtie No alignments
# bowtie No alignments

(Asked by: username_0 on 2021-11-30)

This question was also asked on Biostars
I am working on miRNA alignment using bowtie. But it seems to fail to map. Here are the commands:
bowtie-build -f miRNA.fa miRNA
bowtie --norc -k 1 -v 0 /public/home/wschen_gibh/cpa-seq/ref/miRNA/miRNA -1 SRR16643593_1.NEW.trim.fq -2 SRR16643593_2.NEW.trim.fq -S test.sam 

and here is the result:

and the output sam file:

**Tags:** phylogenetics
> **Comments:**
> **username_1**: Ah, this is "CAP-seq", so there are presumably UMIs in those sequences that you need to deal with first  
> **username_2**: it would help to see the reference file. Can you confirm that you actually expect anything to align to that reference?  

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## 1 answers

### Answer by username_3 on 2021-11-30. Score: 2

You should be using Bowtie 2 instead of bowtie:

Bowtie 1 was released in 2009 and was geared toward aligning the relatively short sequencing reads (up to 25-50 nucleotides) prevalent at the time. Since then, technology has improved both sequencing throughput (more nucleotides produced per sequencer per day) and read length (more nucleotides per read).

http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#how-is-bowtie-2-different-from-bowtie-1
In particular, Bowtie 2 supports local alignment, which is essential when mapping microRNA. All microRNA sequences are short, which means that there will be adapter read-through in the sequenced reads. This adapter sequence will not map to the genome, and that will cause global aligners (like Bowtie) to fail.
An alternative approach would be to trim off palindromic adapter sequences prior to aligning (which it is possible you've tried to do given the file name). Because of adapter read through, standard adapter trimming will not be sufficient. What needs to be trimmed off is the reverse-complement of the adapter sequences. I have no experience using fastp, so don't know if it is capable of doing that.
However, even after trimming there will still be some additional short sequences left on some reads, so a local alignment would still be a good idea.
Doing a local alignment with Bowtie 2 is much easier and faster than trying to troubleshoot adapter read-through issues. The command line you would run is similar, but not identical:
bowtie2-build -f miRNA.fa miRNA
bowtie2 --local -x miRNA -1 SRR16643593_1.NEW.trim.fq -2 SRR16643593_2.NEW.trim.fq | samtools sort > test.bam


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