Abstract 2940: A comparative analysis of library preparation technologies for RNA sequencing from FFPE samples

Abstract Formalin-fixed, paraffin-embedded (FFPE) samples are an invaluable resource in the oncology space, providing access to a vast library of archived diseased tissue samples paired with relevant donor information. Despite the broad utility of these samples, RNA extracted from FFPE tissue is typically difficult to process due to the presence of residual crosslinks and its degraded nature. Further, these samples often vary widely in performance, as the fixation process, block age, block storage, and extraction method can impart large impacts on resulting template quality. As a result, robust and reproducible RNA sequencing from FFPE-derived RNA remains a challenge with unpredictable and high failure rates. We evaluated four commercially available WTA solutions to determine which performed best with this challenging sample type with respect to library complexity, inter-input and intra-sample concordance, and overall reproducibility. Matched fresh frozen and FFPE liver samples were used such that the fresh frozen data set serves as a comparative truth set for the FFPE data. RNA-Seq libraries were prepared from each sample at both 10 ng and 100 ng inputs. The higher input serves as a control while the lower input reflects achievable inputs from very challenging FFPE samples. For 10 ng FFPE-derived RNA, the Watchmaker RNA Library Prep Kit with Polaris Depletion detects more unique genes than other chemistries and has a much higher percentage of genes that overlap with the fresh frozen control libraries. Additionally, differential expression analysis between 10 ng and 100 ng FFPE libraries demonstrates that the Watchmaker solution better preserves the gene expression profile at low inputs, as fewer differentially expressed genes are identified. The Watchmaker chemistry includes a number of features aimed at improving library complexity, including a novel FFPE decrosslinking step, a reverse transcriptase specifically engineered to improve conversion of RNA to cDNA, and fewer bead purification steps to prevent sample loss. The concordance between FF and FFPE samples at variable input masses observed when using the Watchmaker solution promotes a higher degree of confidence when making decisions based on lower quantity and quality samples and enables researchers to access more meaningful biological information. Citation Format: Jennifer Pavlica, Travis Sanders, David Gelagay, Kailee Reed, Thomas Harrison, Giulia Corbet, Josh Haimes, Ariele Hanek, Kristina Giorda, Brian Kudlow. A comparative analysis of library preparation technologies for RNA sequencing from FFPE samples abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2940.