Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA

James C. Willey, Tom B. Morrison, Bradley Austermiller, Erin L. Crawford, Daniel J. Craig, Thomas M. Blomquist, Wendell D. Jones, Aminah Wali, Jennifer S. Lococo, Nathan Haseley, Todd A. Richmond, Natalia Novoradovskaya, Rebecca Kusko, Guangchun Chen, Quan Zhen Li, Donald J. Johann, Ira W. Deveson, Timothy R. Mercer, Leihong Wu, Joshua Xu

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

The 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 will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. To accomplish this, a synthetic internal standard spike-in was designed 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.

Original languageEnglish (US)
Article number100106
JournalCell Reports Methods
Volume1
Issue number7
DOIs
StatePublished - Nov 22 2021
Externally publishedYes

Keywords

  • NGS
  • circulating tumor DNA
  • internal standards
  • liquid biopsy

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Genetics
  • Computer Science Applications
  • Radiology Nuclear Medicine and imaging

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