Abstract 3249: Extending a cfDNA-optimized library preparation workflow to mechanically sheared FFPE DNA for high-quality NGS data

Abstract Formalin-fixed, paraffin-embedded (FFPE) tissues are a foundation of retrospective oncology studies but FFPE samples remain challenging for next-generation sequencing (NGS) due to DNA fragmentation, cross-linking, base damage, and highly variable yield. These factors impact library conversion and complexity and can introduce artifactual variants that confound low-frequency mutation detection. To address similar challenges in liquid biopsy, the Twist cfDNA Library Preparation Kit was engineered to deliver high conversion, high-complexity libraries from low-input cell-free DNA, leveraging an optimized end-repair/ligation chemistry with a Twist-engineered T4 DNA ligase. We demonstrate that this chemistry is well-suited for FFPE DNA that has been mechanically fragmented. Here, we evaluated the performance of the Twist cfDNA Library Preparation Kit on mechanically sheared FFPE control standards as well as FFPE DNA extracted from solid tumor blocks. FFPE samples were processed and extracted, followed by acoustic mechanical shearing. Libraries were prepared with the Twist cfDNA Library Preparation Kit and Twist UMI adapter system followed by target enrichment with oncology-focused capture panels, before sequencing on Illumina instruments. Across FFPE samples of various DIN scores, the cfDNA-optimized workflow produced robust libraries at low input, with high adapter-ligation efficiency, improved library complexity, and uniform target coverage. Duplication rates and off-target reads remained low despite variation in FFPE quality. UMI-based consensus calling efficiently suppressed FFPE-associated artifacts, enabling confident detection of low variant allele frequency (VAF) somatic mutations that align with control expectations. Performance trends scaled favorably with decreasing input mass, supporting sequencing applications for limited, archival material. These results demonstrate that a library preparation chemistry optimized for low-input, fragmented cfDNA can be effectively repurposed for mechanically sheared FFPE DNA. Combining standardized mechanical shearing with the Twist cfDNA Library Preparation Kit and target enrichment provides a unified workflow for liquid biopsy and tissue-based NGS, expanding the utility of Twist’s library preparation and target enrichment solutions for sensitive variant detection in challenging oncology samples. Citation Format: Sean Tighe, Owen Smith, Tiffany Truong, Tong Liu, Elian Lee, Esteban Toro, Siyuan Chen. Extending a cfDNA-optimized library preparation workflow to mechanically sheared FFPE DNA for high-quality NGS data [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 3249.