%0 Journal Article %A Jack Wadden %A Brandon Newell %A Joshua Bugbee %A Robert P. Dickson %A Carl Koschmann %A David Blaauw %A Satish Narayanasamy %A Reetuparna Das %T Ultra-Rapid Somatic Variant Detection via Real-Time Threshold Sequencing %D 2021 %R 10.1101/2021.05.14.444172 %J bioRxiv %P 2021.05.14.444172 %X 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. %U https://www.biorxiv.org/content/biorxiv/early/2021/05/17/2021.05.14.444172.full.pdf