<p>As the other answers/comments have covered, some reads will inevitably align to multiple positions in the genome.
This can come from a variety of sources, like aligning to repetitive regions of the genome, short read fragments (that will likely match multiple regions in the genome, even if it, in theory, came from a single location), and low base call qualities (the aligner doesn't have lots of confidence aligning it it to one location because it could map to another, with some likely mismatched calls in the read).</p>

<p>This isn't inherently a problem, depending on the analysis you want to do after aligning.
In fact, having a single read that is compatible with multiple transcripts is one of the ideas behind the reference index used in <a href="http://www.nature.com/nbt/journal/v34/n5/full/nbt.3519.html" rel="nofollow noreferrer">kallisto</a>, and the "transcript compatibility counts".</p>

<p>If you want to minimize this effect, two easy ways are to create longer reads on your sequencer, or to use paired-end reads (in theory, both ends should align relatively close to each other, so if one of the mates maps to multiple places, you can borrow from the confidence of the alignment of its mate).</p>

CC BY-SA 4.0
2020-06-09T22:27:39.140Z
2020-06-11T14:38:41.833Z
2020-06-11T14:38:41.833Z
James Hawley
<p>You can't always know exactly where every read originated, even if you use long reads to sequence full-length transcripts (odds are there's at least one transcript that's duplicated in the genome). This is also not exactly a problem, such reads can either be discarded or reported as multimapping.</p>

CC BY-SA 4.0
2020-06-09T21:40:44.150Z
2020-06-09T21:40:44.150Z
Devon Ryan
<p>When mapping reads to a reference genome, how is it possible to tell which part of the genome the read is referencing if the genome possibly has the same repeated sequences in different areas? What I mean by this is let's say for the sake of argument that you have a read "AGCTAGCT". If the reference genome has two places where the sequence "AGCTAGCT" is present, how do we tell which one it's coming from? I'm assuming that the length of the read is chosen long enough such that this doesn't happen but perhaps I'm wrong?</p>

CC BY-SA 4.0
2020-06-09T07:26:42.090Z
Of course longer reads will decrease the chance of having such a situation. If you use Bowtie2, you have a randomized search for equal mappings (http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml, see the **Randomness in Bowtie 2** in the manual). In addition, it will report reads that align, 0, 1 or more times.
thomas duge de bernonville
CC BY-SA 4.0
2020-06-09T17:17:09.270Z
@thomasdugedebernonville I'd put that as an answer rather than a comment.
Maximilian Press
<rna-seq><reference-genome><reads>
Clarification about how mapping to a reference genome works in RNA seq
# Clarification about how mapping to a reference genome works in RNA seq

(Asked by: username_0 on 2020-06-09)

When mapping reads to a reference genome, how is it possible to tell which part of the genome the read is referencing if the genome possibly has the same repeated sequences in different areas? What I mean by this is let's say for the sake of argument that you have a read "AGCTAGCT". If the reference genome has two places where the sequence "AGCTAGCT" is present, how do we tell which one it's coming from? I'm assuming that the length of the read is chosen long enough such that this doesn't happen but perhaps I'm wrong?

**Tags:** rna-seq, reference-genome, reads
> **Comments:**
> **username_1**: Of course longer reads will decrease the chance of having such a situation. If you use Bowtie2, you have a randomized search for equal mappings (http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml, see the **Randomness in Bowtie 2** in the manual). In addition, it will report reads that align, 0, 1 or more times.  
> **username_2**: @thomasdugedebernonville I'd put that as an answer rather than a comment.  

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## 2 answers

### Answer by username_3 on 2020-06-09. Score: 1 | Accepted answer

As the other answers/comments have covered, some reads will inevitably align to multiple positions in the genome.
This can come from a variety of sources, like aligning to repetitive regions of the genome, short read fragments (that will likely match multiple regions in the genome, even if it, in theory, came from a single location), and low base call qualities (the aligner doesn't have lots of confidence aligning it it to one location because it could map to another, with some likely mismatched calls in the read).

This isn't inherently a problem, depending on the analysis you want to do after aligning.
In fact, having a single read that is compatible with multiple transcripts is one of the ideas behind the reference index used in [kallisto](http://www.nature.com/nbt/journal/v34/n5/full/nbt.3519.html), and the "transcript compatibility counts".

If you want to minimize this effect, two easy ways are to create longer reads on your sequencer, or to use paired-end reads (in theory, both ends should align relatively close to each other, so if one of the mates maps to multiple places, you can borrow from the confidence of the alignment of its mate).


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### Answer by username_4 on 2020-06-09. Score: 1

You can't always know exactly where every read originated, even if you use long reads to sequence full-length transcripts (odds are there's at least one transcript that's duplicated in the genome). This is also not exactly a problem, such reads can either be discarded or reported as multimapping.


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