Technical Validation of a Next-Generation Sequencing Assay for Detecting Clinically Relevant Levels of Breast Cancer-Related Single-Nucleotide Variants and Copy Number Variants Using Simulated Cell-Free DNA

The Journal of Molecular Diagnostics : JMD
Xin YangJinming Li

Abstract

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 1.25× (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 rob...Continue Reading

Citations

Aug 9, 2019·Journal of Cellular and Molecular Medicine·Puyuan XingYuankai Shi
Jul 6, 2020·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Jing WangZhiyong Liang
Aug 7, 2020·Molecular Genetics & Genomic Medicine·Biao ZhangHaitao Ma
Feb 7, 2019·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Jing WangZhiyong Liang
May 7, 2020·Frontiers in Oncology·Shuhang WangNing Li
Jun 27, 2019·International Journal of Cancer. Journal International Du Cancer·Fei MaBinghe Xu
Nov 13, 2019·Molecular Aspects of Medicine·Edward Geeurickx, An Hendrix
Mar 19, 2021·Frontiers in Immunology·Shuhang WangNing Li

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