1. Advancing Quality-Control for NGS Measurement of Actionable Mutations in Circulating Tumor DNA
- Author
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Thomas Morrison, Tim R. Mercer, Nathan Haseley, Leihong Wu, Jennifer S. LoCoco, Rebecca Kusko, Quan Zhen Li, Wendell D. Jones, Daniel J. Craig, Natalia Novoradovskaya, Joshua Xu, Todd Richmond, Ira W. Deveson, Thomas M. Blomquist, Erin L. Crawford, Aminah Wali, James C. Willey, Brad Austermiller, Donald J. Johann, and Guangchun Chen
- Subjects
Mutation ,Current practice ,Computer science ,Circulating tumor DNA ,medicine ,Variant allele ,Computational biology ,Liquid biopsy ,medicine.disease_cause ,Dna testing ,Precision medicine ,DNA sequencing - Abstract
SUMMARYThe primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample. True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.
- Published
- 2021