Abstract 7139: An optimized extraction and library preparation workflow for sensitive mutation detection in clinical cfDNA samples

Abstract Monitoring cell-free DNA (cfDNA) - small DNA fragments circulating in the bloodstream - provides valuable insights into various tissues throughout the body and diverse health conditions. cfDNA has been particularly useful in tracking cancer progression, although its low abundance can lead to low mutation copy frequencies, especially in early-stage cancers where limited tumor presence yields minimal cfDNA in circulation. Optimizing molecular biology techniques for maximal cfDNA recovery and purity as well as high NGS library conversion efficiency is essential to improve detection of low-frequency cancer mutations. In this study, several cfDNA next generation sequencing (NGS) library preparation workflows were evaluated for sensitivity and workflow efficiency. The NEBNext UltraExpress DNA library prep kit is a new, fast library prep that has not yet been optimized for cfDNA analysis. We compared it with the NEBNext Ultra II DNA Library Prep Kit, which performs well with cfDNA. Both kits were evaluated downstream of the NEAT Liquid Biopsy kit, which is optimized for gDNA removal using Nanotrap Particle technology and results in a highly concentrated DNA elution. With the ability to concentrate down to 20 µL, this extraction kit pairs well with the NEBNext UltraExpress DNA kit which has a 15 µL input. This allows more of the sample to be analyzed, leading to higher detection of low frequency alleles. Initial workflow assessments were analyzed with mutation spike-ins of 0-5% allele frequency in healthy plasma, then confirmed with clinical samples containing known mutations. All workflows produced high complexity libraries with high quality data and successfully detected spike-in mutations, with expected variant allele frequencies. Mutations were also successfully detected in clinical plasma samples demonstrating the essential need for efficient extraction and library prep methods. Citation Format: Lauren P. Saunders, James Erickson, Margaret R. Heider, Jian Sun, Adrian Reich, Ben Lepene. An optimized extraction and library preparation workflow for sensitive mutation detection in clinical cfDNA samples [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 7139.