Accuracy and reproducibility of somatic point mutation calling in clinical-type targeted sequencing data

Treating cancer depends in part on identifying the mutations driving each patient's disease. Many clinical laboratories are adopting high-throughput sequencing for assaying patients' tumours, applying targeted panels to formalin-fixed paraffin-embedded tumour tissues to detect clinically-relevant mutations. While there have been some benchmarking and best practices studies of this scenario, much variant calling work focuses on whole-genome or whole-exome studies, with fresh or fresh-frozen tissue. Thus, definitive guidance on best choices for sequencing platforms, sequencing strategies, and variant calling for clinical variant detection is still being developed.

Because ground truth for clinical specimens is rarely known, we used the well-characterized Coriell cell lines GM12878 and GM12877 to generate data. We prepared samples to mimic as closely as possible clinical biopsies, including formalin fixation and paraffin embedding. We evaluated two well-known targeted sequencing panels, Illumina's TruSight 170 hybrid-capture panel and the amplification-based Oncomine Focus panel. Sequencing was performed on an Illumina NextSeq500 and an Ion Torrent PGM respectively. We performed multiple replicates of each assay, to test reproducibility. Finally, we applied four different freely-available somatic single-nucleotide variant (SNV) callers to the data, along with the vendor-recommended callers for each sequencing platform.

We did not observe major differences in variant calling success within the regions that each panel covers, but there were substantial differences between callers. All had high sensitivity for true SNVs, but numerous and non-overlapping false positives. Overriding certain default parameters to make them consistent between callers substantially reduced discrepancies, but still resulted in high false positive rates. Intersecting results from multiple replicates or from different variant callers eliminated most false positives, while maintaining sensitivity.

Reproducibility and accuracy of targeted clinical sequencing results depend less on sequencing platform and panel than on variability between replicates and downstream bioinformatics. Differences in variant callers' default parameters are a greater influence on algorithm disagreement than other differences between the algorithms. Contrary to typical clinical practice, we recommend employing multiple variant calling pipelines and/or analyzing replicate samples, as this greatly decreases false positive calls.