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bioRxiv · 10.1101/2021.05.14.444172

Ultra-rapid somatic variant detection via real-time threshold sequencing

Wadden, J.·Newell, B.·Bugbee, J.·Dickson, R. P.·Koschmann, C.·Blaauw, D.·Narayanasamy, S.·Das, R.
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Abstract

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. LAMPreys 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.

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Wadden, J., Newell, B., Bugbee, J., Dickson, R. P., Koschmann, C., Blaauw, D., Narayanasamy, S., Das, R.. 2021-05-17. Ultra-rapid somatic variant detection via real-time threshold sequencing. https://doi.org/10.1101/2021.05.14.444172

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