Advancing NGS quality control to enable 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 (NG...

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Bibliographic Details
Published in:Cell reports methods 2021-11, Vol.1 (7), p.100106, Article 100106
Main Authors: Willey, James C., Morrison, Tom B., Austermiller, Bradley, Crawford, Erin L., Craig, Daniel J., Blomquist, Thomas M., Jones, Wendell D., Wali, Aminah, Lococo, Jennifer S., Haseley, Nathan, Richmond, Todd A., Novoradovskaya, Natalia, Kusko, Rebecca, Chen, Guangchun, Li, Quan-Zhen, Johann, Donald J., Deveson, Ira W., Mercer, Timothy R., Wu, Leihong, Xu, Joshua
Format: Article
Language:English
Subjects:
circulating tumor DNA
internal standards
liquid biopsy
NGS
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Summary: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. [Display omitted] •Synthetic spike-in IS control for technical errors in NGS•IS enable measurement of true-positive mutations not detected by current practices•IS included in hybrid capture NGS libraries do not interfere with existing workflows•IS may be used as an orthogonal quality control for UMI or non-UMI library analysis Despite an increasing demand for precision medicine enabled by NGS measurement of actionable mutations in circulating tumor DNA (ctDNA) specimens, the ability to reliably measure and report low-frequency mutations using current NGS practices is limited. Challenges include low- or poor-quality specimens and technical errors that vary among samples and mutation sites. Here, we designed synthetic internal standards (IS) and methods for their use to better control for technical error in NGS in assessment of ctDNA specimens. The goal was to determine whether this would improve quality control, resulting in increased clinical sensitivity without loss of specificity. Willey et al. spiked synthetic internal standards (IS) into contrived circulating tumor DNA samples to control for technical error in NGS. IS enabled calculation of technical error rate and limit of detection for each actionable mutation in each sample, increasing the number of measurable true positives without loss of specificity.
ISSN:2667-2375
2667-2375
DOI:10.1016/j.crmeth.2021.100106