Question: Hi all,

I have three somatic variants (mutations) detected in a single tumour sample. They come from Whole Genome Sequencing technologies and to call them normal and tumour samples were compared.

The mutations are supported by the same reads. It's a bit suspicious to me because this could indicate that the reads are from another part of the genome.

I have been searching a LOT for online tutorials about manual inspection of mutations with IGV. But I only found a couple of them that are interesting. However, I could not identify if these three mutations are real or not:

[Dropbox link to IGV screenshot of variants.][1]

[TinyPic link to IGV screenshot of variants.][2]

a) The quality of the reads is good: MAPQ between 60 and 29.

b) The variants are supported by reads from both strands.

c) There are no germline mutations annotated in dbSNP, 1000 genomes, etc.

d) Blat scores for the supporting reads in other regions of the genome are lower than 300, while the scores for this region are higher than 900.

But as I said before, I think that the fact that they are on the same reads is not a good signal.

Do you think that these could be false mutations? or real mutations from another region of the genome?

Thanks in advance.

[1]: https://www.dropbox.com/s/8sh8xf4koan9l3w/d_e.png?dl=0
[2]: http://es.tinypic.com/r/24pjqzl/9

Answer: >The mutations are supported by the same reads. It's a bit suspicious to me because this could indicate that the reads are from another part of the genome.

It could also indicate that there are three adjacent mutations, or another sort of mutation. Without additional context, is it difficult to determine whether the reads are true variants. In addition to the properties you have identified, you should consider:

prior somatic mutation: are any of the SNVs in COSMIC?

strand bias: looks fine as there are reads supporting the variant originating from both strands

allele frequency: the variants appear to be have somewhere around 20-40% BAF. Is this consistent with the BAF of upstream and downstream SNV?

sequence context/alignment artifacts: do the reads actually represent three nearby SNV? Have a look at the sequence context of the mutations. Depending on the flanking sequence there may be a more parsimonious explanation. For example, in some sequencing contexts, the 3 SNVs could be explained by 1bp insertion that the aligner prefers to align as 3 SNVs. Are the reads containing the variants soft clipped? Is this aactually a STR repeat expansion/contraction?

normal coverage: is there sufficient coverage in the normal for the variants to be reliably called as somatic? (the SNV caller should calculate this)

kataegis: do these mutations occur in a region of kataegis?

variant quality score: what quality score does the variant caller assign to these variants?

SV: is the tumour highly structurally rearranged? Could these be explained by a single DNA repair event? More generally, any explanation that results in the simultaneous generation of the mutations increases the plausibility of the SNVs as a single event explanation removes your implicit assumption that somatic SNVs should not be clustered so closely.

> But as I said before, I think that the fact that they are on the same reads is not a good signal.

It just means they are phased together and all occurred on the same chromatid. It does not necessarily make them less likely - especially if they can be explained by a single event. Events such as chromothripsis result in hundreds of structural rearrangements from the same chromatid.

TLDR: from the limited information available, they look plausible.