Journal
JOURNAL OF MOLECULAR DIAGNOSTICS
Volume 19, Issue 4, Pages 525-536Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jmoldx.2017.04.007
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Funding
- National Population and Family Planning Commission of the People's Republic of China Special Fund for Health Scientific Research in the Public Interest [201402018]
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Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency >= 0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25x(25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as Low as 0.5% for SNVs and copy number of 1.25x for CNVs.
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