Jang Y et al., The American Journal of Human Genetics - Every cell in the body picks up its own small set of DNA changes over a lifetime, and many of these somatic mutations are carried by only a tiny fraction of cells. This study, part of the SMaHT Network, asks how reliably standard bulk whole-genome sequencing can find mutations present at a variant allele fraction below 1%. The team built a ground truth from one donor's skin: clonal stem cell lines grown from single fibroblasts defined real mutations, and lines from other skin biopsies of the same donor defined mutations that should be absent. With about 1,360× Illumina coverage confirmed by PacBio and AVITI, point mutations below 1% were detected accurately in the easily mapped parts of the genome. Key terms: somatic mosaicism, whole-genome sequencing, variant allele fraction, benchmarking, SMaHT.

Study Highlights:
Primary fibroblasts from a 29-year-old donor contained five subclones, captured as clonal iPSC lines, giving benchmark mutations from 0.1% to 25% variant allele fraction, while iPSC lines from other biopsies of the same donor supplied negative controls; both sets held about 11,000 SNVs and 2,800 indels. Using the negative controls, PacBio and AVITI had the lowest substitution error rates in easy regions, 0.003% and 0.005%, and mismatch rates rose by 1 to 2 orders of magnitude in harder regions. In fibroblasts, more than 99.5% of bulk-only calls above 1% were confirmed by PacBio or AVITI, the estimated false-positive rate was near zero above 0.8% and about 11% below 0.5%, and homopolymer indels could be detected reliably only above 3%. In postmortem lung and colon, combining about 200× from two tissues found 13 additional shared mutations, a 145% gain in sensitivity below 3%, all confirmed by PacBio or single-cell data.

Conclusion:
Ultra-deep bulk whole-genome sequencing can detect point mutations below 1% allele fraction with high accuracy, provided calling is restricted to easily mapped regions and sample quality is checked, and pooling coverage across tissues from one donor recovers shared developmental mutations. The benchmark comes from a single donor's fibroblasts, confirmation below 0.5% was limited by long-read and AVITI coverage, most indels sit in homopolymers or hard regions where detection remains unreliable, and postmortem samples needed extra filtering.

Article title:
Accurate detection of sub-1% frequency somatic mutations by whole-genome sequencing

First author:
Jang Y

Journal:
The American Journal of Human Genetics

DOI:
10.1016/j.ajhg.2026.09.010

Reference:
Jang Y., Gardiner S., Wang Y., Natu A., Dehankar M.K., Tomasini L., et al. Accurate detection of sub-1% frequency somatic mutations by whole-genome sequencing. The American Journal of Human Genetics. (2026). https://doi.org/10.1016/j.ajhg.2026.09.010

License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/

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