Ultra-rapid somatic variant detection via real-time targeted amplicon sequencing
Open Access
- 15 July 2022
- journal article
- research article
- Published by Springer Science and Business Media LLC in Communications Biology
- Vol. 5 (1), 1-12
- https://doi.org/10.1038/s42003-022-03657-6
Abstract
Molecular markers are essential for cancer diagnosis, clinical trial enrollment, and some surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform due to time-consuming DNA extraction, targeted amplification, and library preparation times. In this work, we present a proof-of-principle approach for sub-1 hour targeted variant detection using real-time DNA sequencers. By modifying existing protocols, optimizing for diagnostic time-to-result, we demonstrate confirmation of a hot-spot mutation from tumor tissue in ~52 minutes. To further reduce time, we explore rapid, targeted Loop-mediated Isothermal Amplification (LAMP) and design a bioinformatics tool—LAMPrey—to process sequenced LAMP product. LAMPrey’s concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Using LAMPrey, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.Funding Information
- National Science Foundation (1652294)
- U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (R01-NS124607, R01-NS119231)
- U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke
- U.S. Department of Defense (CA201129P1)
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