LAUSR

Clinical oncology heavily relies on formalin-fixed, paraffin-embedded (FFPE) tissue samples for histology and molecular characterization. The chemical and physical modifications of nucleic acids introduced during fixation, storage and purification negatively impact molecular profiling and vary from sample-to-sample. Conventional sonication and ligation-based library preparation is considered the gold-standard approach for FFPE samples, but it is time-consuming and expensive. Importantly, these processes introduce artifacts that impact downstream analysis and interpretation (Haile et al., 2019). We have developed a novel fragmentation chemistry that virtually eliminates hairpin artifacts, achieving levels on par with non-FFPE control samples. Our fragmentation method is highly scalable, exhibits minimal sequence bias, and reduces cost and workflow inefficiencies associated with mechanical shearing. In this study, we developed a unified method for library preparation from FFPE samples which produces similar insert sizes across variable input mass and quality of FFPE samples. We carefully evaluated the performance of this method relative to a sonication-based approach employing the KAPA HyperPrep kit. Libraries were constructed from 50 to 200 ng of FFPE DNA, inputs typically used for NGS, ranging from low-to high-quality as assessed using a qPCR-based method and DNA integrity (DIN). Targeted sequencing was performed using a 37 kb custom oncology hybridization capture panel to investigate molecular complexity. Our workflow virtually eliminated hairpin artifacts that were present in up to 4.5% of reads in sonication-based libraries. Soft-clipping was also 3- to 7-fold lower in libraries prepared with the Watchmaker kit relative to sonicated DNA libraries, improving overall sequencing economy. Furthermore, the mean target coverage achieved with the Watchmaker kit was comparable to or higher than sonication libraries using the same input mass. Because input masses were normalized post-shearing, which typically results in 20-40% sample loss, coverage with our approach is significantly higher relative to sonication, if normalizing to pre-sheared DNA input. Watchmaker DNA Library Preparation with Fragmentation enables high-quality DNA library preparation from damaged FFPE samples, producing high target coverage, uniform insert size, and minimizing sequencing artifacts to improve sensitivity and specificity. This approach is highly scalable and automatable, enabling various oncology applications. Citation Format: Giulia Corbet, Philip Benson, Kailee Reed, Skyler Mishkin, Thomas Harrison, Kristin Scott, Zane Jaafar, Kristina Giorda, Josh Haimes, Martin Ranik, Brian Kudlow. A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 215.