Details

Autor(en) / Beteiligte

• Yang, Xin
• Chu, Yuxing
• Zhang, Rui
• Han, Yanxi
• Zhang, Lucheng
• Fu, Yu
• Li, Dan
• Peng, Rongxue
• Li, Dongdong
• Ding, Jiansheng
• Li, Ziyang
• Zhao, Meiru
• Zhang, Kuo
• Lu, Tian
• Yi, Lang
• Wu, Qisheng
• Lin, Guigao
• Xie, Jiehong
• Liu, Tao
• Yang, Ling
• Yi, Xin
• Li, Jinming

Titel

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

Ist Teil von

• The Journal of molecular diagnostics : JMD, 2017-07, Vol.19 (4), p.525-536

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2017

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.