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bioRxiv · 10.64898/2026.04.08.717310

Evaluation of somatic variant calling methods on high coverage tumour-only amplicon sequencing data in a clinical environment

Bharne, D.·Gaston, D.
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Abstract

One of the current workhorses of next-generation sequencing in clinical molecular diagnostics laboratories for profiling somatic mutations in tumours are amplicon-based targeted sequencing panels. Many open-source somatic variant callers are available; however, their use in clinical applications remains under explored. Therefore, we integrated outputs of six variant callers (FreeBayes, MuTect2, Pisces, Platypus, VarDict and VarScan) into a Snakemake pipeline and evaluated tumour-only data from the HD789 commercial reference standard sequenced in triplicate on three different sequencing runs using the Illumina AmpliSeq Focus panel on MiSeq and NextSeq 2000. A 1:4 dilution sample was sequenced for evaluating limits of variant detection. The called variants were analysed along depth, allele frequency, and other sequencing metrics. The variant callers were evaluated by their level of concordance and performance on known somatic variants. FreeBayes consistently called the largest number of somatic variants in each sample but also included more potential artifacts. Overall, FreeBayes, VarScan, MuTect2, and Pisces had the best performance on HD789 data.

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Bharne, D., Gaston, D.. 2026-04-11. Evaluation of somatic variant calling methods on high coverage tumour-only amplicon sequencing data in a clinical environment. https://doi.org/10.64898/2026.04.08.717310

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