Abstract 4086: A streamlined and modular workflow solution enhances coverage uniformity and achieves high library complexity from genomic DNA/total RNA using both short and long read sequencers

Abstract The application of NGS-based assays can be limited by the availability and quality of genomic DNA from certain sample sources. Practical factors such as turnaround time, workflow simplicity, target enrichment capability, and overall performance are crucial in determining whether NGS is the preferred method for germline or somatic mutation detection. In addition, due to certain short read sequencer limitations (e.g., mapping rate, repetitive regions, etc.), enabling long read capture and sequencing allows for better detection of structural variations, complex rearrangements, and variants in highly repetitive or polymorphic regions. To address these challenges, we have developed a flexible and automation-compatible workflow solution that supports both WGS (PCR-free and PCR-based, depending on the application) and target enrichment sequencing. Our modular workflow accommodates a wide range of DNA and RNA inputs (10-500 ng), standard and in-line molecular-barcoded adaptors, enzymatic and mechanical shearing methods, fast or overnight probe hybridization, and up to 384 unique dual sample indices to eliminate index hopping. Our enzymatic library preparation method combines fragmentation, end repair, and dA-tailing in a single step, allowing the use of samples with low concentrations. We demonstrate high base coverage uniformity and minimal bias across all GC content (Fold 80 of 1.12-1.13) using three bacteria of varying GC content (30%-67%) and human gDNA. Finally, our novel fast hybridization buffer formulation provides maximum complexity while maintaining high uniformity and base coverage, specifically addressing the issue of complexity loss when multiple libraries are pooled during bait hybridization. As we demonstrate, with small changes in the protocol, efficient long read capture (>5kb) and sequencing can be successfully accomplished. For Research Use Only. Not for use in Diagnostic procedures. Citation Format: Bahram Arezi, Adam Janssen, Nikki Liu, Sergey Shiryaev, Keith Chen, Bilan Hsue, Brandyn Clark, Jeff fox, Nedda Saremi, Inlora Jingga, Kyeong-soo Jeong, Khine Win, Gilbert Amparo, Neelima Mehendale, Jayati Ghosh, Cindy Hon. A streamlined and modular workflow solution enhances coverage uniformity and achieves high library complexity from genomic DNA/total RNA using both short and long read sequencers [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 4086.