Ultra-rapid somatic variant detection via real-time targeted amplicon sequencing

Area of Science:

• Molecular Biology
• Bioinformatics
• Oncology

Background:

• Molecular markers are crucial for cancer diagnosis, clinical trial enrollment, and surgical decisions.
• Current sequencing-based variant detection methods are time-consuming, often taking hours due to extensive sample preparation.
• There is a critical need for ultra-rapid, intraoperative variant detection to improve patient management.

Purpose of the Study:

• To develop and validate a proof-of-principle approach for sub-1-hour targeted variant detection using real-time DNA sequencers.
• To explore the use of rapid, targeted Loop-mediated Isothermal Amplification (LAMP) for faster DNA amplification.
• To design and implement a bioinformatics tool (LAMPrey) for efficient processing of LAMP sequencing data.

Main Methods:

• Modification of existing protocols and optimization for rapid time-to-result in DNA sequencing.
• Implementation of targeted Loop-mediated Isothermal Amplification (LAMP) for rapid DNA amplification.
• Development of LAMPrey, a bioinformatics tool with a concatemer-aware alignment algorithm for processing LAMP sequencing data.

Main Results:

• Demonstrated confirmation of a hot-spot mutation from tumor tissue in approximately 52 minutes using modified sequencing protocols.
• Achieved confirmation of a hot-spot mutation (with 250x support) from tumor tissue in under 30 minutes using LAMP and LAMPrey.
• LAMPrey's alignment algorithm enhanced the recovery of diagnostically relevant information, enabling more rapid detection compared to standard methods.

Conclusions:

• The presented approach significantly reduces the time required for targeted variant detection, enabling intraoperative analysis.
• Loop-mediated Isothermal Amplification (LAMP) coupled with the LAMPrey bioinformatics tool offers a promising strategy for ultra-rapid molecular diagnostics.
• This methodology has the potential to revolutionize cancer diagnosis and intraoperative decision-making by providing near real-time genetic information.