This webinar introduces Roche's new Kappa Hyper PET (Primer Extension Target Enrichment) technology, a novel method for target enrichment in next-generation sequencing workflows. Kappa Hyper PET combines the high performance of hybrid capture with the speed and ease of amplicon-based workflows, enabling an automatable, single-day process. The technology is designed for the detection of all major variant classes, including single nucleotide variants (SNVs), insertions/deletions (indels), copy number variants (CNVs), microsatellite instability (MSI), and known/novel fusion transcripts. The presentation details the methodology, which involves biotinylated target-specific primers, single-cycle extension, bead-based capture, and a secondary primer release mechanism, followed by post-capture PCR. Performance data is presented for somatic tissue DNA, cell-free DNA, and somatic tissue RNA workflows using pan-cancer and lung cancer fusion panels, demonstrating high on-target rates, superior coverage uniformity compared to anchored multiplex methods, and robust variant detection across various sample qualities and input amounts.
Exploring Kappa Hyper PET: A Novel Target Enrichment Technology
0:02 Introduction to Kappa Hyper PET: Kappa Hyper PET (Primer Extension Target Enrichment) is a new target enrichment technology from Roche. It uses shotgun libraries from DNA or RNA as input.
0:42 Key Advantages: It combines the high performance of hybrid capture with the ease of use and rapid turnaround time of amplicon workflows, offering an automatable, single-day workflow.
0:57 Variant Class Detection: Capable of detecting single nucleotide variants (SNVs), short insertions/deletions (indels), copy number variants (CNVs), microsatellite instability (MSI), and known/novel fusion transcripts.
1:36 Library Preparation: Begins with a shotgun library, ideally with Kappa unique dual index adapters, with unique molecular identifiers (UMIs) for somatic mutation calling.
2:30 Capture Primer: A target-specific, biotinylated primer anneals to the target molecule.
2:40 Extension: A single cycle of PCR extends the primer through the rest of the library molecule, creating a probe.
3:00 Bead Binding: Strep Avidin beads bind to the biotin-labeled extension product, capturing the target.
3:08 Wash Step: Off-target molecules are washed away, leaving only on-target molecules.
3:23 Second Primer Release: A second target-specific primer binds upstream of the initial extension product, extends through it, and releases the target molecules into solution. This unique step increases the on-target rate.
4:01 Post-Capture PCR: Illumina P5 and P7 amplification primers are used to enrich the sample, preparing it for QC, pooling, sequencing, and analysis.
4:19 Fast Turnaround Time: Hyper PET offers significantly faster turnaround times (e.g., ~5 hours total for RNA workflow) compared to hybrid capture methods (which typically involve overnight hybridization). It offers similar workflow times to anchored multiplex methods.
4:53 Efficiency for Long Regions: Hyper PET can process fewer samples than typical amplicon technologies when covering long contiguous regions.
5:10 Workflow Time Variability: The total turnaround time varies based on the library preparation method (e.g., somatic tissue DNA workflow, cell-free DNA workflow, somatic tissue RNA workflow), but the enrichment step itself is consistent across all applications.
6:19 Superior Coverage Uniformity: Compared to anchored multiplex methods, Hyper PET demonstrates superior performance and excellent coverage uniformity across target regions, as shown in an IGV view of the APC gene.
Used the Hyper Pan Cancer panel (CNV and MSI calling not enabled for cfDNA).
12:08 Enrichment Performance: On-target rates ranged from 67-74%. Increasing read depth significantly improved coverage metrics (e.g., % panel exon bases >3000X improved from 73% to 95% with increased read pairs for 50ng input).
13:27 Variant Detection: 100% of short variants at 1% allele frequency were detected. 88.2-94.1% of short variants at 0.5% allele frequency were detected (undetected variants due to configurable threshold settings).
Utilized a Hyper PET Lung Cancer Fusion panel (16 oncogenes, 137 exons, detects known/novel fusions) including 4 housekeeping genes as controls.
16:26 Enrichment Performance: After ribosomal RNA removal, on-target rates were 92-97% across all samples, input amounts, qualities, and types. Housekeeping targets comprised 40-60% of reads.
17:03 Variant Detection: 100% of fusions in reference cell line RNA samples were detected.
17:24 Key Takeaways: The Hyper PET technology leverages Roche's design expertise for challenging genomic regions, saves precious samples, improves sensitivity with no panel splitting, offers superior sequencing efficiency and uniformity, and is optimized for somatic variant detection from various sample types.