Abstract 6530: Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs
Authors: Crary-Dooley Florence K. , Xi Liu , van der Merwe Ruben , Furtado Nitya M. , Li Jingchuan , Lin Junyan , Ochoa Manuel , Godwin Brian 
Published: April 2023

Abstract
Background: The presence of circulating tumor DNA (ctDNA) can be used for minimal residual disease (MRD) monitoring and guide adjuvant therapy decisions. However, even when present, total ctDNA could be less than 0.01% of cell free DNA (cfDNA). Sequencing such low mutation frequency can be costly due to high sequencing read depth and accuracy requirements. The ability to enrich for low-abundance variants could improve detection performance and greatly lower sequencing cost by reducing the sequencing depth required.
Purpose: The KAPA HyperPETE (Primer Extension Target Enrichment) workflow employs primer extension reactions to specifically capture and release target library molecules for sequencing, detects all major somatic variant types (SNV, InDel, CNV, MSI status, and novel fusion transcript partners) and could be leveraged to specifically target certain alleles. In this study, we assessed the variant enrichment capabilities of the KAPA HyperPETE workflow.
Methods: Variant specific primers were designed to 8 low allele frequency single nucleotide variants (SNVs) in mixed cell line samples (NA12878 and NA24143, two Epstein-Barr virus transformed B cell female cell lines, with variant allele frequency (VAF) 1%, 0.5%, and 0.1%). Variant enrichment primers were used alongside a 37kb panel in the KAPA HyperPETE workflow. Additional optimizations and a study enriching all low allele frequency SNVs* in SeraCare ctDNA Mutation Mix v2 reference samples (VAF 1%, 0.5%, and 0.125%) are planned.
Results: The preliminary results exhibited a median of 249✕, 159✕, and 86.5✕ enrichment of the targeted variants compared to the expected VAFs of 0.1%, 0.5%, and 1%, respectively. All 8 targeted variants were detected at 100K, 150K, and 550K read pairs per variant for VAF 1%, 0.5%, and 0.1%, respectively. Results from further optimizations and the study using SeraCare ctDNA samples will be shared at the AACR meeting.
Conclusion: Preferential enrichment of specific variants down to 0.1% can be achieved using the KAPA HyperPETE Workflow. Utilizing primers designed for variant enrichment, we simultaneously enriched multiple variants and demonstrated the potential to minimize required sequencing. The KAPA HyperPETE workflow can be a powerful, cost-effective tool for detecting low frequency mutations in MRD research applications.
KAPA HYPERPETE is For Research Use Only. Not for use in diagnostic procedures. KAPA HYPERPETE is a trademark of Roche.
*Except GNA11 (COSM52969) due to stretches of polybase. Seraseq ctDNA Mutation Mix v2 gene list available on SeraCare website.
Citation Format: Florence K. Crary-Dooley, Liu Xi, Ruben van der Merwe, Nitya M. Furtado, Jingchuan Li, Junyan Lin, Manuel Ochoa, Brian Godwin. Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6530.