Abstract 4070: Demonstrating analytical performance of Agilent Avida Duo utilizing novel multiomic reference standards

Abstract Measuring both genetic and epigenetic status by target enrichment NGS workflows is critical for high-sensitivity oncology applications using cell-free samples (cfDNA). Conventional workflows accomplish this by splitting a sample into multiple tests, potentially limiting sensitivity. The Agilent Avida target enrichment technology enables high-efficiency enrichment of native DNA fragments, preserving epigenetic markers such as methylation. This method, termed Avida Duo, measures both mutation and methylation status, maximizing the information that can be obtained from any single cfDNA sample. Multiomic assays have become increasingly important in research applications and determining their analytical performance is key to ensure they are fit for purpose. Currently, standards with known variants or methylation levels are available, however, there is a lack of multiomic standards for evaluation of more advanced dual modalities. We utilized commercially available mutation and methylation standards to create a combined reference to assess the performance of the Avida Duo workflow. To create this reference, the mutation status of the methylation-specific reference and the methylation profile of the mutation-specific reference was determined. We blended these fully characterized references in defined ratios to achieve known variant allele frequencies and methylation levels similar to real-world cfDNA samples. Using this combined reference standard, we demonstrated the analytical performance of the Avida Duo technology across an input range of 5-20ng using a 55.2kb mutational panel that combines hotspot and full-gene enrichment and a 173.9kb methyl panel that covering relevant differentially methylated regions (DMRs). From this, we were able to demonstrate robust detection of variants down to <0.5% VAF and 1% methylation of DMRs. The Avida Duo technology offers a single workflow for a combined interrogation of genetic and epigenetic information of cfDNA and as such, requires new tools to appropriately characterize the performance. Here we demonstrate an approach for generating reference samples for assay development, QC, and benchmarking the analytical performance of multiomic assays. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Michael Ruvolo, Yingmin Wang, Grace Zhao, Heng Wang, Kyeong-Soo Jeong, Yun Bao, Shengrong Lin, Bram Herman, Mistuni Ghosh. Demonstrating analytical performance of Agilent Avida Duo utilizing novel multiomic reference standards [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 4070.