PT - JOURNAL ARTICLE AU - Jack Wadden AU - Brandon Newell AU - Joshua Bugbee AU - Robert P. Dickson AU - Carl Koschmann AU - David Blaauw AU - Satish Narayanasamy AU - Reetuparna Das TI - Ultra-Rapid Somatic Variant Detection via Real-Time Threshold Sequencing AID - 10.1101/2021.05.14.444172 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.05.14.444172 4099 - http://biorxiv.org/content/early/2021/05/17/2021.05.14.444172.short 4100 - http://biorxiv.org/content/early/2021/05/17/2021.05.14.444172.full AB - Molecular markers are becoming increasingly important for cancer diagnosis, proper clinical trial enrollment, and even surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform. In this work, we present Threshold Sequencing, a methodology for designing protocols for targeted variant detection on real-time sequencers with a minimal time to result. Threshold Sequencing analytically identifies a time-optimal threshold to stop target amplification and begin sequencing. To further reduce diagnostic time, we explore targeted Loop-mediated Isothermal Amplification (LAMP) and design a LAMP-specific 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. Coupled with time-optimized DNA extraction and library preparation, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.Competing Interest StatementThe authors have declared no competing interest.