Authors: ENitya Furtado¹*, Florence Crary-Dooley¹*, Mingyang Cai¹*, Brian Godwin¹, Liu Xi¹, Jimmy Tu¹, Liesl Joubert², Marius Snyman², Mariana Fitarelli-Kiehl³, Spencer Debenport³, and Jingchuan Li¹
¹ Roche Sequencing Solutions, Pleasanton, CA 94588, ² Roche Sequencing Solutions, Cape Town, South Africa 7925, ³ Roche Sequencing & Life Science, Wilmington, MA, USA, *co-first authors
Abstract: KAPAHyperPETE (Primer Extension Target Enrichment) is a novel target enrichment solution newly launched by Roche, which employs primer extension reactions to specifically capture and release target library molecules for sequencing. It offers the simplicity and speed of amplicon-based workflows and the high performance of hybridization-based capture. KAPA HyperPETE is a single day workflow that demonstrates high coverage uniformity and detects all major somatic variant types (SNV, InDel, CNV, MSI status, and novel fusion transcript partners).
Pre-capture multiplex pooling is an established approach in capture-based target enrichment methods to decrease the number of sample reaction tubes handled during enrichment steps. While pre-capture multiplex pooling reduces the reagent cost and hands-on time when processing many samples, it is understood that enrichment performance can be impacted. These performance losses are usually minimal when detecting heterozygous and homozygous SNPs or InDels while using high quality genomic DNA, but can potentially be problematic when detecting somatic variants of less than 5% allele frequency. This can be compounded by the use of low quality and/or low nucleic acid input amounts commonly derived from oncology samples.
We optimized pre-capture multiplexing conditions for oncology samples in the Tissue DNA, Tissue RNA, and Plasma cfDNA enrichment workflows. The results demonstrate sequencing performance metrics and variant calling results comparable to single-plex capture. KAPA HyperPETE Workflow with pre-capture multiplexing improves turnaround time, optimizes cost, and increases the number of samples that can be processed simultaneously while retaining high performance.
The KAPA HyperPETE pre-capture multiplexing is not an officially supported protocol yet in somatic oncology research applications.