Hi,
We sequenced genomes of ancestors/unevolved and evolved populations of a bacterial species. We would like to determine genomic variants (SNPs/small INDELs) of the evolved strains in comparison to the ancestor. We expect that the ancestor is clonal, while the evolved populatioin contains a mixture of different polymorphisms. The ancestor is >99% identical to the reference genome on NCBI.
For variant calling I see 3 options:
1. Calling of variants based on the NCBI reference and removal of those variants that also occured in the ancestor.
2. Mapping of the variants from the ancestor to the NCBI reference, modify the reference according to the ancestor and use the result as new reference sequence for mapping of evolved populations.
3. Using a somatic variant calling tool to compare variants between the ancestor and the evolved population directly.
I think that the first two methods may be problematic, because they do only consider variants that were called positive. Information about low quality variants gets lost and little differences in predictions for the same variants may cause problems.
I was wondering if somatic variant calling could be a better alternative, because it directly considers the difference between two sequencing datasets. Information on application of this method is usually limited to eukaryotic datasets, mostly cancer vs. tumor cells. I did not find any information on the possibility of application for experimental evolution in bacterial datasets.
Does anyone has a suggestions for how to proceed and if somatic variant calling would be an appropriate method?
Best,
Chris
I don't see a reason why somatic SNP calling should not work in your case. However, if your subclonality is vast, your mutations might be buried in sequencing errors/artifacts. Sequencing errors are in the range of 0.1-1%, so if one mutation in a subclone is "diluted" by other subclones, you will likely miss those, or not be able to discern between the variants and the background sequencing error.
If you are searching for a somatic SNP caller I#d recommend Mutect2 (https://software.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walkers_cancer_m2_MuTect2.php).
Best,
Peter
One advantage of the second approach over the first one is a slightly increased mapping rate. Therefore, I would not recommend to use the first approach.
However, the third approach is even better. Due to differences between variant callers, it might be useful to compare and combine the results of several tools. GATK should definitely be among them.
Moreover, it might be interesting to look at the allele frequency of interesting variants. Beneficial (causal) mutations should be more frequent than neutral ones. This is at least the case if your 'evolved' means that a certain selection pressure was applied.
Best,
Boas
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