<p>I am trying to identify somatic mutations on healthy subjects (specifically in WES samples). As of now, I have found a couple of papers who have done similar studies such as <a href="https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1919-5" rel="nofollow noreferrer">this paper</a> where somatic mutations in healthy subjects are identified (using RNA-SEQ data). But in my case, I want to find somatic mutations from normal WES samples. For somatic mutations, we need both tumors and matched normal samples to identify somatic mutations but how to identify somatic mutations from normal samples only. Please suggest. Thanks.</p>

CC BY-SA 4.0
2020-09-08T12:21:17.640Z
You can follow the [GATK pipeline](https://gatk.broadinstitute.org/hc/en-us/articles/360035894731-Somatic-short-variant-discovery-SNVs-Indels-)
csgroen
CC BY-SA 4.0
2020-09-08T12:32:20.833Z
@csgroen: As I have mentioned in my question, I have to find somatic mutations in normal samples only. The link you have shared contains the GATK pipeline for somatic mutations with tumor and matched normal samples. I am already aware of this. Thanks.
Lot_to_learn
CC BY-SA 4.0
2020-09-08T12:41:24.203Z
Hm, I see... The issue is that you need your 'baseline' germline genome to be able to call somatic mutations. Otherwise, how can you know if they are germline or somatic?
csgroen
CC BY-SA 4.0
2020-09-08T13:08:27.763Z
Yes. Precisely. Or I just found one way in this paper (Whole-genome DNA sequencing provides an atlas of somatic mutagenesis in healthy
human cells and identifies a tumor-prone cell type) in which conditions are not clear to me.
Lot_to_learn
CC BY-SA 4.0
2020-09-08T13:12:25.997Z
From a quick read, my interpretation is that they aligned against a reference genome (hg19), then removed annotated known germline SNPs (using dbSNP as reference)
csgroen
CC BY-SA 4.0
2020-09-08T17:19:59.260Z
check out this one:https://gatk.broadinstitute.org/hc/en-us/articles/360035535932-Germline-short-variant-discovery-SNPs-Indels-, this is for germline mutations
Phoenix Mu
CC BY-SA 4.0
2020-09-09T08:32:18.463Z
@PhoenixMu he wants somatic, not germline mutations. That's actually the challenge, as it is normal tissue and doesn't have a proper reference. But personally, I think what I described from my interpretation of the paper you shared seems to be at least on the correct path. Align against reference genome and remove common SNPs. It's not perfect, there will be false postiives and negatives, but it should work overall.
csgroen
CC BY-SA 4.0
2020-09-09T09:35:30.993Z
@csgroen: Yes. I agree with you. But my problem is the result's accuracy. The pipeline used in the above-mentioned paper considered 3 variant callers and allowing only variants with low allele fraction. How low value of allele fraction gives a better solution?? And 3rd condition "variants that occurred during the in vitro culture of
the clone" is also unclear to me.
Lot_to_learn
CC BY-SA 4.0
2020-09-09T09:54:44.950Z
Paper quote: "To eliminate all other germline variants, including rare ones, we utilized the low confidence germline variants called by GTEx. [...] Our goal was to exclude as many germline variants as possible, so we reasoned that using all germline variants called by GTEx—including the low-quality ones—was the safest option to minimize germline mutation contamination in our somatic mutation calls." I believe they removed all annotated variants, even if they were low confidence. I don't understand your in vitro comment though, makes no sense for this study.
csgroen
<mutations><exome><somatic>
Somatic mutations for normal WES samples
# Somatic mutations for normal WES samples

(Asked by: username_0 on 2020-09-08)

I am trying to identify somatic mutations on healthy subjects (specifically in WES samples). As of now, I have found a couple of papers who have done similar studies such as [this paper](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1919-5) where somatic mutations in healthy subjects are identified (using RNA-SEQ data). But in my case, I want to find somatic mutations from normal WES samples. For somatic mutations, we need both tumors and matched normal samples to identify somatic mutations but how to identify somatic mutations from normal samples only. Please suggest. Thanks.

**Tags:** mutations, exome, somatic
> **Comments:**
> **username_1**: You can follow the [GATK pipeline](https://gatk.broadinstitute.org/hc/en-us/articles/360035894731-Somatic-short-variant-discovery-SNVs-Indels-)  
> **username_0**: @csgroen: As I have mentioned in my question, I have to find somatic mutations in normal samples only. The link you have shared contains the GATK pipeline for somatic mutations with tumor and matched normal samples. I am already aware of this. Thanks.  
> **username_1**: Hm, I see... The issue is that you need your 'baseline' germline genome to be able to call somatic mutations. Otherwise, how can you know if they are germline or somatic?  
> **username_0**: Yes. Precisely. Or I just found one way in this paper (Whole-genome DNA sequencing provides an atlas of somatic mutagenesis in healthy human cells and identifies a tumor-prone cell type) in which conditions are not clear to me.  
> **username_1**: From a quick read, my interpretation is that they aligned against a reference genome (hg19), then removed annotated known germline SNPs (using dbSNP as reference)  
> **username_2**: check out this one:https://gatk.broadinstitute.org/hc/en-us/articles/360035535932-Germline-short-variant-discovery-SNPs-Indels-, this is for germline mutations  
> **username_1**: @PhoenixMu he wants somatic, not germline mutations. That's actually the challenge, as it is normal tissue and doesn't have a proper reference. But personally, I think what I described from my interpretation of the paper you shared seems to be at least on the correct path. Align against reference genome and remove common SNPs. It's not perfect, there will be false postiives and negatives, but it should work overall.  
> **username_0**: @csgroen: Yes. I agree with you. But my problem is the result's accuracy. The pipeline used in the above-mentioned paper considered 3 variant callers and allowing only variants with low allele fraction. How low value of allele fraction gives a better solution?? And 3rd condition "variants that occurred during the in vitro culture of the clone" is also unclear to me.  
> **username_1**: Paper quote: "To eliminate all other germline variants, including rare ones, we utilized the low confidence germline variants called by GTEx. [...] Our goal was to exclude as many germline variants as possible, so we reasoned that using all germline variants called by GTEx—including the low-quality ones—was the safest option to minimize germline mutation contamination in our somatic mutation calls." I believe they removed all annotated variants, even if they were low confidence. I don't understand your in vitro comment though, makes no sense for this study.  

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