Abstract 6597: Optimization of DNA and RNA extraction from tumor FFFPE samples and end-to-end automation and analytical validation of the TSO 500 HT v1 library preparation

Abstract Formalin-fixed paraffin-embedded (FFPE) techniques are cost-effective, long-term preservation methods for clinical samples, enabling next-generation sequencing (NGS) for expanded discovery and diagnostic applications. DNA and RNA extraction and library preparation must be optimized to ensure efficient downstream NGS analysis. We first focused on optimizing the quality and quantity of DNA and RNA recovery from FFPE samples representing various oncology conditions, using the truXTRAC® FFPE Tissue Extraction Kits (Covaris) automated on the Kingfisher Flex instrument (Thermo). We then analyzed the extracted DNA and RNA from FFPE tumor samples, including colorectal, kidney, breast, lung, gastric, and endometrial cancer, along with control samples from Horizon Oncospan and Seraseq, for analytical validation of the TSO 500HT v1 assay. RNA inputs were tested at 20ng, 40ng, and 80ng, while DNA inputs were tested at 20ng, 40ng, and 100ng. The TSO 500HT assay targets 523 genes to assess all DNA and RNA variant types, including SNV, CNV, indels, fusions, MSI, and TMB. For library preparation, 72-sample batches were processed using manual and fully automated workflows, with the automation protocol executed on the Biomek i7 (Beckman Coulter). Sequencing was performed on the Illumina NovaSeq 6000 and NovaSeq X Plus, using a 2x100bp paired-end configuration to generate approximately 50 million DNA reads and 8-10 million RNA reads per library. This approach demonstrated an overall inter-run and intra-run variant detection accuracy of over 90%, indicating highly repeatable and reproducible results. Variability between runs, lots, and operators was ≥99%. Our analysis showed high assay performance, with ≥96% of samples passing Illumina's cut-off metrics, particularly with 40ng DNA and 80ng RNA inputs. The analytical sensitivity for the TSO 500HT assay was benchmarked with ≥95% sensitivity for SNVs, ≥85% for InDels, ≥90% for CNVs, ≥90% for MSI, and ≥90% for TMB. These results provide a high-throughput, automated solution for dual DNA and RNA extraction from FFPE samples, demonstrating a robust, reproducible, and highly accurate targeted sequencing method for variant detection in oncology patients. Citation Format: Nripesh Prasad, Supriya Singh, Rebecca Beatty, Rachel Rock, Melanie Robinson, Annamaria Szanto, Sara Hildebrand, Boris Umylny, Thomas Halsey. Optimization of DNA and RNA extraction from tumor FFFPE samples and end-to-end automation and analytical validation of the TSO 500 HT v1 library preparation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6597.