Advancing quality-control for NGS measurement of actionable mutations in circulating tumor DNA

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 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. ### Competing Interest Statement JCW has 5-10% equity interest in and serves as a consultant to AccuGenomics, Inc. Technology relevant to this manuscript was developed and patented by JCW, ELC, and TB, and is licensed to AccuGenomics, Inc. These relationships do not alter our adherence to all policies on sharing data and materials. The views presented in this article do not necessarily reflect the current or future opinion or policy of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification and not intended as an endorsement.