Abstract 121: Single stranded ligation enhances the performance of the duet evoC 6-base assay, enhancing value in low DNA input applications including liquid biopsy.

Abstract The 6-base genome, provided by duet evoC, provides the canonical 4-base genome whilst simultaneously distinguishing 5-methylcytosine from 5-hydroxymethylcytosine to provide the 5th and 6th, epigenetic, bases. The 6-base genome has been shown to be a powerful tool for the discovery of biomarkers of early biological change and provides mechanistic insight across important applications including oncology, liquid biopsy and neurology. In these applications it is important to maximise the information derived from precious DNA samples, both in terms of multiomic data and material recovery. [RO1] Here we present the next evolution of the duet product portfolio. Through implementation of a novel single-stranded ligation approach, we demonstrate marked improvements[RC2] in assay performance including material recovery and DNA methylation calling sensitivity, whilst maintaining high genetic and epigenetic accuracy. These improvements derive from the ability of single-stranded ligation to maintain the original cfDNA strands. In contrast, end-repair can fill-in 5’ overhangs with unmodified cytosines (reducing methylation detection sensitivity) and can introduce errors at 5’ overhangs in addition to nicks and gaps. In addition, end-repair can remove information by degrading 3’ overhangs. The single-stranded ligation method also captures both single and double stranded cfDNA, maximizing recovery of unique molecules. This is a critical parameter for liquid biopsy applications, where it is important to assay as many circulating-tumor (ctDNA) molecules as possible. We anticipate that these improvements will further enhance the power of the duet portfolio to reveal powerful novel biomarkers and provide mechanistic insight in sample-constrained liquid biopsy applications. Citation Format: Tom Charlesworth, Audrey Vandomme, Jens Fullgrabe, Ermira Lleshi, Lidia Prieto-Lafuente, Edyta Bocian-Canepa, Elena Pahiti, Rebecca Ellwood, Mengjie Li, Marjana Lila, Robert Crawford, Robert J. Osborne. Single stranded ligation enhances the performance of the duet evoC 6-base assay, enhancing value in low DNA input applications including liquid biopsy [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 121.