Demonstrate performance of a complete automation and reagent workflow for analysis of cfDNA from bodily fluids. The efficient extraction of cfDNA from bodily fluids is a unique challenge due to the very low concentrations of nucleic acid. The extraction process along with library preparation is a laborious workflow, where human variability can lead to increased variability in the downstream analysis. Integrated DNA Technology (IDT) and Beckman Coulter (BC) have teamed up to provide a complete automation and reagent workflow for analysis of low frequency variants in cfDNA. The Apostle MiniMax™ High Efficiency Isolation Kit from BC provides complex, utilized magnetic nanoparticles to effectively capture cfDNA. IDT's library prep kit utilizes novel chemistry to maximize conversion, suppress adapter-dimer formation, reduce chimera rates, and facilitate double strand consensus analysis to call ultra-low frequency variants. Finally, IDT's xGen™ hybrid capture products maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. The Biomek i5 and i7 Hybrid workstations bring out the best performance from these reagents. The Biomek NGS workstations protocol is written with a modular design with safe stop points, making it customizable for each lab. The automated protocol uses Beckman's Demonstrated Method Interface tools which include: Biomek Method Launcher to run the method without going into Biomek software, Method Options Selector to choose the run parameters with a user friendly interface, Guided labware Setup to set the deck with labware based on the run parameters, DeckOptix Final Check software to help reduce deck setup errors. We demonstrate the performance of this complete workflow with a range of plasma inputs (4-8 mL). Using control samples with known variant frequencies, the workflow yields high library complexity, 100% positive predictive value, and reliable detection of <0.5% mutant allele frequency variants. With real cfDNA, the workflow demonstrates both high cfDNA and sequencing library yields along with high library complexity. The combination of these reagents on the Biomek workstations provides a robust and reproducible solution for the analysis of cfDNA.

Citation Format: Nicole Roseman, Shilpa Parakh, Hsiao-Yun Huang, Kevin Lai, Timothy Barnes, Lyn Lewis, Ushati Das Chakravarty, Anastasia Potts, Alisa Jackson, Amy Yoder, Jessica Sheu, Tzu-Chun Chen. Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5863.