KAPA HyperPETE Choice  and KAPA HyperPETE Explore

KAPA HyperPETE Choice and KAPA HyperPETE Explore Panels

Overview

KAPA HyperPETE Panels for custom regions

Roche introduces its target enrichment custom panels, KAPA HyperPETE Choice Panels and KAPA HyperPETE Explore Panels, for DNA or RNA. KAPA HyperPETE Choice Panels and KAPA HyperPETE Explore Panels enable custom designs up to 250Kb for DNA inputs and 50Kb for RNA inputs.

Features and Benefits of KAPA HyperPETE Custom Panels

  • Better by design, using Roche's renowned oligo design and selection algorithms, enable access to challenging genomic regions
  • Seamless online generated designs in the HyperDesign Tool either with a few clicks or through an expert designer-assisted process
  • High uniformity for better sequencing efficiency with less optimization
  • Validated secondary analysis solution available by NAVIFY® Mutation Caller.


Product highlights

KAPA HyperPETE Custom Panels. Better by design.

  • Quickly and efficiently cover your custom regions with the high-performing KAPA HyperPETE Custom Panels

    • Rely on Roche’s proven design and primer selection expertise to improve your desired target coverage and maximize the data return from your research. Use the HyperDesign Tool to prepare your custom design in four (4) simple steps, include an optimized MSI (Microsatellite Instability) target set into your design or consult with Roche’s expert designers team for your special design needs.

  • Take fewer steps to optimal performance and sequencing efficiency

    • Experience high performance from your very first panel design iteration as a result of Roche’s design expertise and extensive KAPA HyperPETE Workflow optimization.

  • Achieve confident variant detection for all major mutation classes
    • Detect Single Nucleotide Variants (SNVs), insertions or deletions (Indels), Copy Number Variants (CNVs), Fusions and Microsatellite Instability (MSI) in your regions of interest, using an optimized workflow with integrated secondary analysis by the NAVIFY® Mutation Caller.
       

 

Tissue DNA (FFPET): Variant Detection and Performance Data

Figure 1. Performance metrics using tissue DNA (FFPET). High specificity with deep and broad target coverage were demonstrated using a KAPA HyperPETE Choice Panel (capture target of 88 Kb plus 25 Kb of the Roche preset MSI targets). Percent of reads on-target ranged from 77.1% to 87.7% (median 84.4%), percent of bases covered at > 300X ranged from 97.2% to 98.7% (median 98.4%), and percent of bases covered at > 500X ranged from 94.4% to 98.3% (median 97.4%).

Eight (8) FFPET DNA samples and eight (8) control FFPET DNA samples of varying quality were tested in duplicates by following the KAPA HyperPETE Workflow for Somatic Tissue DNA Preparation. High quality DNA samples had a normalized Q score > 0.4 with input range of 20 ng to 34.4 ng, medium quality > 0.22 with input range of 37 ng to 55.5 ng, and low quality > 0.09 with input range of 62.6 ng to 121.1 ng. Final libraries were sequenced on an Illumina NextSeq™ 550 system. Total read pairs (2x150 bp) per sample ranged from 12.5M to 20.4M and data was analyzed using NAVIFY® Mutation Caller to assess enrichment and variant detection performance.

Table 1 (a, b, c, d). Highly confident detection of SNVs, Indels, CNV and MSI using Horizon Discovery FFPET control and CrownBio FFPET xenograft samples. Performance was demonstrated using a KAPA HyperPETE Choice Panel (capture target of 88 Kb plus 25 Kb of the Roche preset MSI targets). True positive detection rates were 100% for SNVs, or Indels (Table 1a), CNVs (Table 1b) and MSI (Table 1c), without any false negatives. False positive rate was 0% for microsatellite stable samples (Table 1d). Final libraries were sequenced on an Illumina NextSeq™ 550 system. Total read pairs (2x150 bp) per sample ranged from 12.5M to 20.4M and data was analyzed using NAVIFY® Mutation Caller to assess the variant detection.

Table 1a. Performance in SNV and Indel detection
Sample Input (ng) Expected allele frequency Total replicates Expected variants False negatives True Positive Rate
HD200 20 5% 4 24 SNVs 0 100%
HD200 20 5% 4 4 Indels 0 100%
HD789 20 5% 4 8 Indels 0 100%
Table 1b. Performance in CNV detection
Sample Input (ng) Gene Expected copies Total replicates False negatives True Positive Rate
HD789 20 MET 4.5 4 0 100%
Table 1c. Performance in MSI detection (true positive rate)
Sample (xenografts) Input (ng) Expected status Total replicates Reported status (≥40% = MSI) True Positive Rate
DU145 62.6 - 343.3 MSI 2 MSI: 83.1% - 84.3% 100%
SW48 41.3 - 45.7 MSI 2 MSI: 97.1% - 98.3% 100%
Table 1d. Performance in MSI detection (false positive rate)
Sample (xenografts) Input (ng) Expected status Total replicates Reported status (<40% = MSS) False Positive Rate
BT474 40.3 - 51.7 MSS (Microsatellite stable) 2 MSI: 23.4% - 29.2% 0%
MDA-MB-453 72.5 MSS (Microsatellite stable) 1 MSI: 28.5% 0%

 

Tissue RNA (FFPET): Fusion detection and performance data

KAPA HyperPETE Choice RNA Panel enables confident fusion detection from RNA samples

  • Single-day from RNA to the sequencer, optimized and low hands-on time workflow
  • Sequencing efficiency by high on-target rates
  • Confident fusion detection with high true positive rates
  • KAPA HyperPETE technology allows detection of unknown fusion partners 

Figure 2. Performance using tissue RNA (FFPET).  High specificity was demonstrated by the high percent of reads on-target when starting from 10 ng of various quality (low, medium, high) input FFPE RNA. On-target rate was 94.6% to 97.9% (includes housekeeping and fusion targets) with good performance across all sample input qualities, and types. The on-target rate was calculated following rRNA read removal (0.5% to 3.5% of all reads). One reference cell-line sample (in duplicates), 3 reference FFPET samples (in duplicates) and 8 normal adjacent FFPET samples were used to assess performance.

RNA was extracted with the Roche High Pure FFPET RNA Isolation Kit and quality was determined with the DV200 score using the Agilent RNA 6000 Pico Assay on the Bioanalyzer. The KAPA HyperPETE Workflow for Tissue RNA Fusion Transcript Preparation using a KAPA HyperPETE Choice RNA Panel (capture target size ~13 Kb) was followed. Libraries were generated using the KAPA RNA HyperPrep kit in combination with the KAPA Universal UMI Adapters and 10ng of RNA while adjusting PCR cycles based on the DV200 score. Libraries were captured using the KAPA HyperPETE Reagent Kit and sequenced on an Illumina NextSeq™ 550 system. Total read pairs (2x150 bp) per sample ranged from 2.4M to 3.5M, and data was analyzed using NAVIFY® Mutation Caller to assess enrichment and variant detection performance.

Table 2. Fusion detection performance using tissue RNA (FFPET). All fusions (100%) were detected in the reference cell line sample at 10 ng RNA input amount. The data of the reference cell-line was used to assess fusion detection performance. Data analysis was performed using the NAVIFY® Mutation Caller.

Variant RNA Input Amount (ng) Total replicates Expected Fusions True Positive Rate
Fusion
10 2 6 100%