Ultra-Rapid Somatic Variant Detection via Real-Time Threshold Sequencing

Molecular markers are becoming increasingly important for cancer diagnosis, proper clinical trial enrollment, and even surgical decision making, motivating ultra-rapid, intraoperative molecular diagnostics. Sequencing-based diagnostics are considered the gold standard approach, but typically take hours to perform. In this work, we present Threshold Sequencing, a methodology for designing sequencing-based diagnostic protocols 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 diagnosis versus standard read alignment approaches. Coupled with time-optimized DNA extraction and library preparation, we demonstrate confirmation of a hot-spot mutation (250x depth) from tumor tissue in less than 30 minutes.