Abstract 6599: A comprehensive benchmark study of multiple library amplification solutions for NGS

Abstract The emerging field of precision oncology utilizes next-generation sequencing (NGS) to guide decisions based on individual genetic signatures. As demand for these applications grows, minimizing errors and biases introduced during library preparation and sequencing is critical. Advances in sequencing chemistries have improved base quality scores, shifting the focus to library preparation to improve data quality. PCR amplification is a key potential source of error during NGS library preparation; single-base substitutions, insertions, and deletions (indels) introduced by PCR in addition to PCR-induced biases in DNA length and GC content can distort data leading to decreased assay sensitivity and specificity. Improved library amplification, combined with recent innovations in sequencer technology, will increase the reliability of NGS assays and lead to better assay performance. Herein, we benchmark several commercial PCR amplification systems, including two solutions from Watchmaker Genomics: Equinox, and a re-engineered library amplification mix in development. We employed a UMI-powered NGS assay to quantify GC-uniformity, chimeric reads, base incorporation fidelity, and indel rates in microsatellite sequences. Libraries were sequenced on multiple platforms to leverage the ultra-high base quality scores of recent sequencing chemistries, resulting in low-noise measurements of polymerase fidelity. We also interrogate UMI family depth uniformity, a powerful proxy for template sequence bias and an informative indicator for error-correction efficacy. Furthermore, we assess amplification size bias by comparing the ability to amplify long templates in the presence of short templates. Compatibility with both SPRI and streptavidin beads was assessed. Finally, we test the ability to generate high terminal library yield, which is important for hybridization capture workflows. These results indicate that while some products may excel in one or two metrics, they dramatically underperform in others. Equinox, in particular the newly engineered variant, demonstrates robust performance across all relevant metrics, making it a leading solution for precise and unbiased library amplification. Citation Format: Giulia Corbet, Caitlyn Mendik, Lee French, Leo Karamanof, Meghan Oddy, Andrew Sutherland, Philip Benson, Josh Haimes, Ross Wadsworth, Travis Sanders, Craig Marshall, Kristina Giorda, Thomas Harrison, Eduard Casas, Brian Kudlow. A comprehensive benchmark study of multiple library amplification solutions for NGS [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 6599.