KAPA HyperPETE Custom Probes

Roche’s proven design and primer selection expertise can be utilized to improve the desired target coverage and maximize the data return from research. Users can use the HyperDesign Tool to prepare a custom design in four simple steps, include an optimized MSI (Microsatellite Instability) target set into the design or consult with Roche’s expert designers team for any special design needs.

Figure 1: Tissue DNA (FFPET)—Variant Detection and Performance Data.

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 with an internal Roche secondary analysis pipeline to assess enrichment and variant detection performance.

Figure 2: Tissue RNA (FFPET)—Fusion detection and performance data.

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 with an internal Roche secondary analysis pipeline to assess enrichment and variant detection performance.

KAPA HyperPETE Custom Probes can detect Single Nucleotide Variants (SNVs), insertions or deletions (Indels), Copy Number Variants (CNVs), Fusions and Microsatellite Instability (MSI) in regions of interest, using an optimized workflow.