Abstract 3244: Rapid, one-tube sub 10-minute whole genome library prep for cfDNA

Abstract As next-generation sequencing (NGS) costs have decreased, it is increasingly more feasible to perform whole genome sequencing instead of a targeted approach to obtain more comprehensive genome information, better cover difficult regions, and provide insights for rare diseases. With increasing adoption of NGS, there is an emphasis on optimizing and streamlining the entire process, particularly library preparation and sequencing steps to reduce turnaround times and simplify workflows. Standard library prep has many complex and time-consuming steps such as end-repair, A-tailing, and PCR that increase barrier to entry for users, require specialized equipment, and can also induce unique errors to the final library. Additionally, each of these steps can also have impacts to the efficiency in which you convert input material into sequenceable library which may be critically limiting in material-limited applications. We have developed a new methodology for a rapid, one-tube, whole genome library prep prior to sequencing on the AVITI ™ platform. Through DNA fragmentation and single-adapter addition, this approach allows for highly efficient PCR-free library creation in under 10 minutes of total time, while maintaining the ability for paired-end sequencing chemistry through circularization on-instrument. We demonstrate the library prep workflow’s efficiency on extracted gDNA (Coriell and E. coli, H. influenzae, and R. palustris bacteria to span content across GC ranges) and Horizon cfDNA material (Mimix Multiplex I cfDNA reference at 1-5% VAF). We found high conversion efficiency with <80ng of gDNA allowing for full AVITI24 ™ output of up to 1.5B reads, with high estimated library complexity. Additionally, when more depth per target is needed, we show compatibility of the fast, clean-up free workflow with our Trinity ™ technology which removes need for off-board washing and QC steps, allowing for library prep and enrichment within the timeframe of a standard NGS library prep (<2 hours). These techniques hold promise to streamline and integrate NGS preparation and sequencing while maintaining quality and reducing workflow burden. Citation Format: Ben Krajacich, Seana Lymer, Kevin Green, Xiaodong Qi, Kyle Donohoe, June (Junhua) Zhao, Michael Previte. Rapid, one-tube sub 10-minute whole genome library prep for cfDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3244.