Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma

Journal of Cancer
Donglai LiuChuntao Zhang

Abstract

Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor (EGFR) gene at serial allelic frequencies. All reference materials were quantified and validated via droplet digital polymerase chain reaction (ddPCR), and then were distributed to eight domestic manufacturers for the collaborative evaluation of the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton and BGISEQ). Results: All expected mutations except one at extremely low allelic frequencies were detected, despite some differences in coefficient of variation (CV) which increased with the decrease of allelic frequency ...Continue Reading

Methods Mentioned

BETA
nucleic acid extraction
PCR

Software Mentioned

SigmaPlot
GraphPad Prism
SPSS

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