<p>If you want to look at a few, specific reads manually, you can do this on <a href="https://varsome.com/" rel="nofollow noreferrer">VarSome</a>. We have recently released a new feature that allows you to enter single NGS or Sanger reads and get the list of variants detected in that read. For example, <a href="https://varsome.com/single-read/hg38/AGTCCRAGTTGTAAATGGTACACTCGGCGTAAGCCTGAAAAGATAAAATCAAAGATGTAAAGGTGAGCACAGTCTAAGTTCTCTCTGAAGTGTCAATGGGAATGCAGATTGGATTAAATAAATGCTGCCCAAGTGCATACTCAAAGAGGC" rel="nofollow noreferrer">here</a>:</p>
<p><a href="https://i.sstatic.net/pawMJ.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/pawMJ.png" alt="screenshot of VarSome showing an aligned read" /></a></p>
<p>In this example, there was one variant identified when comparing to the human hg38 genome assembly:</p>
<ul>
<li><a href="https://varsome.com/variant/hg38/CYP24A1(NM_000782.5):c.1186C%3ET" rel="nofollow noreferrer">CYP24A1(NM_000782.5):c.1186C&gt;T</a> which is at chr20:54158136 G⇒A (note that the CYP24A1 gene is on the - strand which is why the C to T variant in HGVS notation is a G to A in the genome). You can then click on the variant link(s) to investigate the variant's effect and annotation.</li>
</ul>
<p>Obviously, this isn't practical for many reads, but it can be handy if you just want to look at a few manually. More importantly, NGS is very error prone (which is why we sequence at high depth), and you should absolutely not assume that just because a variant is present in a single read it was also actually present in the source DNA you sequenced.</p>
<hr />
<p>Disclaimer: I work for the company that builds VarSome.</p>

CC BY-SA 4.0
2022-03-12T13:37:30.597Z
2022-03-13T12:07:05.563Z
2022-03-13T12:07:05.563Z
terdon
<p>After sequencing (Illumina) of some DNA, I generated a sam file through alignment of a fastq file (using Bowtie2). Instead of using variant calling programs, I want to know the specific variances, compared to the reference genome, in each specific read.</p>
<p>For example, I'm only interested in C to A mutations. I would like to include reads that contain MORE than 5 C to A mutations. Afterwards I would like to analyze the specific sequence context of that mutation and for doing that I need to know the 3 nucleotides before and after the C to A mutation, which gives a context of NNNCNNN.</p>
<p>I looked at samtools mpileup or bcftools, but I can't find the exact thing that I want to analyze. Any ideas?</p>
<p>Thank you!</p>

<sam><samtools><variant-calling>
Detect mutation context in a read of a sam file
# Detect mutation context in a read of a sam file

(Asked by: username_0 on 2022-03-11)

After sequencing (Illumina) of some DNA, I generated a sam file through alignment of a fastq file (using Bowtie2). Instead of using variant calling programs, I want to know the specific variances, compared to the reference genome, in each specific read.

For example, I'm only interested in C to A mutations. I would like to include reads that contain MORE than 5 C to A mutations. Afterwards I would like to analyze the specific sequence context of that mutation and for doing that I need to know the 3 nucleotides before and after the C to A mutation, which gives a context of NNNCNNN.

I looked at samtools mpileup or bcftools, but I can't find the exact thing that I want to analyze. Any ideas?

Thank you!

**Tags:** sam, samtools, variant-calling

---

## 1 answers

### Answer by username_1 on 2022-03-12. Score: 1

If you want to look at a few, specific reads manually, you can do this on [VarSome](https://varsome.com/). We have recently released a new feature that allows you to enter single NGS or Sanger reads and get the list of variants detected in that read. For example, [here](https://varsome.com/single-read/hg38/AGTCCRAGTTGTAAATGGTACACTCGGCGTAAGCCTGAAAAGATAAAATCAAAGATGTAAAGGTGAGCACAGTCTAAGTTCTCTCTGAAGTGTCAATGGGAATGCAGATTGGATTAAATAAATGCTGCCCAAGTGCATACTCAAAGAGGC):

[![screenshot of VarSome showing an aligned read](https://i.sstatic.net/pawMJ.png)](https://i.sstatic.net/pawMJ.png)

In this example, there was one variant identified when comparing to the human hg38 genome assembly:

* [CYP24A1(NM\_000782.5):c.1186C>T](https://varsome.com/variant/hg38/CYP24A1(NM_000782.5):c.1186C%3ET) which is at chr20:54158136 G⇒A (note that the CYP24A1 gene is on the - strand which is why the C to T variant in HGVS notation is a G to A in the genome). You can then click on the variant link(s) to investigate the variant's effect and annotation.

Obviously, this isn't practical for many reads, but it can be handy if you just want to look at a few manually. More importantly, NGS is very error prone (which is why we sequence at high depth), and you should absolutely not assume that just because a variant is present in a single read it was also actually present in the source DNA you sequenced.

---

Disclaimer: I work for the company that builds VarSome.


---

