Abstract
Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.
Highlights
Generation sequencing (NGS) is a powerful technology used in clinical genetic testing for the diagnosis of cancer and inherited diseases [1,2,3]
We performed dual deep sequencing to investigate the reproducibility of each step, such as DNA amplification and variant calling
We provide a practical Next generation sequencing (NGS)-based somatic variant calling workflow applicable to cancer gene panel testing, which is advantageous for more accurate somatic mutation detection
Summary
Generation sequencing (NGS) is a powerful technology used in clinical genetic testing for the diagnosis of cancer and inherited diseases [1,2,3]. Cancer gene panel testing is the first line of examination for clinical cancer diagnosis and is a time-saving and cost-effective method involved in cancer treatment. During the DNA amplification step of NGS library preparation, de novo low-frequency nucleotide sequence alternations are generated by DNA polymerase errors, even when using high-fidelity enzymes to amplify the targets [7,8,9]. PCR polymerase errors affect the detection of low-frequency variants and accurate measurement of allele frequency [10,11,12,13]. Previous studies have compared the error rates of several DNA polymerases for generating heterozygous and homozygous variants but there have been no studies that have examined the detection and measurement of allele frequency for de novo low-frequency variants [14]
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