Abstract 6725: Introducing new technology for high performance whole exome sequencing with unparalleled hybridization time flexibility and capability for low to high capture-input

Abstract Whole exome sequencing (WES) via hybridization capture is a targeted sequencing approach to specifically enrich the protein-coding regions of the human genome. The human exome comprises ∼1% of the entire genome but contains the majority of relevant variants for researchers. Targeting this region saves on sequencing costs and data storage while also allowing greater sequencing depth of regions of interest. However, current hybridization capture workflows are challenging to execute in the laboratory due to high-temperature sample and reagent handling, pre-heating of multiple buffers, long hybridization times, and narrow library input ranges. Widening the input range is desirable because it can allow for fewer PCR cycles in library preparation or enable high multiplexing during hybridization capture, the latter of which can lower reagent costs. To evaluate targeted sequencing performance across a wide range of sample inputs and hybridization times, we used low to high total input—1-plex 100ng to 12-plex 6000ng—and hybridized for as short as 1 hour up to overnight. We used the Integrated DNA Technologies (IDT) novel xGen™ Hybridization and Wash v3 kit, which features a user-friendly workflow without hot-liquid handling or temperature-sensitive urgency. This performance is benchmarked side-by-side with a current commercial hybridization-capture kit. Using these methods, we achieve similar targeting, coverage, uniformity, and GC-balance across all samples with minimal impact from low inputs and short hybs. Further, we calculated the reference/alternate (ref/alt) allelic fraction and found no considerable differences between capture conditions, indicating the potential of this workflow for genotyping applications with low-input samples and/or short hybridizations. This flexibility in hybridization time and library input along with the streamlined workflow allows researchers to meet diverse experimental demands and effectively capture the human exome. Citation Format: Daniel Saatman, Jinglie Zhou, Ekaterina Star, Roshani Codipilly, Alexandra Wang. Introducing new technology for high performance whole exome sequencing with unparalleled hybridization time flexibility and capability for low to high capture-input [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 6725.