Overview

Features and Benefits of KAPA HyperPETE Catalog Panels

  • Better by design, using Roche’s renowned content and panel design expertise
  • High uniformity for better sequencing efficiency with less optimization
  • Readily available for fast delivery
  • Validated NAVIFY® Mutation Caller secondary analysis solution available.

Product highlights

KAPA HyperPETE Catalog Panels enrich with efficiency from cfDNA samples

  • Increase your sequencing efficiency with high on-target rates
  • Confidently detect all major variants with broad target coverage at high depth
  • Elevate your detection accuracy using the KAPA Universal UMI Adapter

Plasma cfDNA: Variant Detection and Performance Data

Figure 1. Performance metrics using plasma and reference cell-line cfDNA. High specificity with deep and broad unique target coverage were demonstrated using the KAPA HyperPETE Workflow for Somatic Plasma cfDNA Preparation, across all sample input amounts and types. On-target rate was 65% - 73% for the KAPA HyperPETE Hot Spot Panel and 70% - 73% for the KAPA HyperPETE Pan Cancer Panel. At 50 ng cfDNA input the percent of panel exon bases covered at ≥ 1000X of unique depth (PCR duplicates removed) was > 99% for the KAPA HyperPETE Hot Spot Panel and 97% - 98% for the KAPA HyperPETE Pan Cancer Panel. The respective percentages at ≥ 3000X unique depth were 80% - 94% and 67% - 85%. At 10 ng of cfDNA input the unique depth was concordant with the available unique genome equivalents (~3300 in 10 ng of DNA) and the percentages at ≥ 1000X unique depth were 83% - 99% and 80% - 94% per panel respectively.

Libraries were generated using the KAPA HyperPrep Kit and the KAPA Universal UMI Adapter from either 10 ng or 50 ng of plasma cfDNA or fragmented reference cell-line DNA as input, and individually captured using the KAPA HyperPETE Reagent Kit and the respective panel. Final libraries were sequenced on an Illumina NextSeq™ 550 System with 6M - 14M (median: 8M) high-quality read pairs (2x150 bp) allocated per sample for the KAPA HyperPETE Hot Spot Panel, and 55M - 81M (median: 66M) high-quality read pairs allocated per sample for the KAPA HyperPETE Pan Cancer Panel. Two (2) Seraseq® ctDNA Mutation Mix samples in duplicates and 16 healthy donor plasma cfDNA samples were used to assess the performance. Data was analyzed using the NAVIFY® Mutation Caller.

Table 1. Variant detection performance using reference cell-line cfDNA. High true positive detection rates were demonstrated for short variants (Single Nucleotide Variants - SNVs, and Indels) at low variant allele expected frequencies across all reference cell line samples (2 Seraseq® ctDNA Mutation Mix samples in duplicates). From both input amounts (10 ng and 50 ng) 100% of short variants (SNVs and Indels) were detected at 1% allele frequency using either of the two KAPA HyperPETE Panels. At 0.5% allele frequency from 10 ng input, the true positive detection rate was 94% and 96.9% using the KAPA HyperPETE Hot Spot Panel and the KAPA HyperPETE Pan Cancer Panel, respectively. At 0.5% allele frequency from 50 ng input, the true positive detection rate was 100% and 98.4% using the KAPA HyperPETE Hot Spot Panel and the KAPA HyperPETE Pan Cancer Panel, respectively.

Panel Input (ng) Allele Frequency Expected Variants Detected Variants True Positive Rate



KAPA HyperPETE Hot Spot Panel
10
1.0%

56
56 100%
50 56 100%
10
0.5%

50
47* 94%
50 50 100%



KAPA HyperPETE Pan Cancer Panel
10
1.0%

70
70 100%
5 70 100%
10
0.5%

64
62* 96.9%
50 63* 98.4%

* Variants were present but not included in the true positive rate calculation as read support was lower than the cutoff used in the analysis pipeline.

KAPA HyperPETE Catalog Panels enrich with efficiency from FFPET DNA samples

  • Increase your sequencing efficiency with high on-target rates
  • Confidently detect all major variants with broad target coverage at high depth
  • Increase your success and save precious DNA samples, adjusting your input amount by using the KAPA NGS FFPE DNA QC Kit 
  • Elevate your detection accuracy using the KAPA Universal UMI Adapter

Tissue DNA (FFPET): Variant detection and performance data

Figure 2. Performance metrics using FFPET DNA. High specificity with deep and broad unique target coverage were demonstrated using the KAPA HyperPETE Workflow for Somatic Tissue DNA Preparation with the KAPA HyperPETE Pan Cancer Panel, across all sample input amounts and types. A median of 73% of reads on-target was achieved. From quality adjusted input, greater than 98% and greater than 93% of exon bases were covered at ≥ 300X and ≥ 500X unique depth, respectively. From 50 ng of input the low quality DNA performed slightly lower than medium and high quality DNA by achieving greater than 92% and greater than 75% of exon bases covered at ≥ 300X and ≥ 500X unique depth, respectively.  As expected, when using 10 ng DNA input amount the performance was variable and in general lower, particularly at ≥ 500X unique depth.

FFPET DNA was extracted using the KAPA NGS DNA Extraction Kit and qualified using the KAPA NGS FFPE DNA QC Kit. Libraries were generated following the KAPA HyperPETE Workflow for Somatic Tissue DNA Preparation. For quality adjusted input 60 ng - 248 ng of low quality, 40 ng - 54 ng of medium quality and 20 ng - 26 ng of cell-line and high quality FFPET DNA were used. Quality scores were calculated according to the KAPA HyperPETE Workflow for Somatic Tissue DNA Preparation. High quality DNA samples had a normalized Q score ≥ 0.5, medium quality ≥ 0.22 and low quality ≥ 0.04. High quality total read pairs (2x150 bp) of an Illumina NextSeq™ 550 System ranged from 19M - 55M (median: 35M) per sample. Sixteen (16) adjacent normal FFPET samples, four (4) CrownBio xenograft FFPET samples (in duplicates), two (2) Horizon Discovery reference FFPET samples (in duplicates) and twelve (12) Horizon Discovery cell-line reference samples (in duplicates) were tested. Data was analyzed using the NAVIFY® Mutation Caller.

Table 2 (a, b, c). Variant detection performance using tissue DNA. High, 100% true positive detection rates were demonstrated for short variants (SNVs and Indels, table 2a) at an expected frequency of ~ 5%, CNVs (Copy number variants, table 2b) at an expected copy number of ~4.5 - 6 and MSI (Microsatellite instability, table 2c). The true positive detection rate was assessed across the subset of the tested samples with known variants (cell-lines, FFPET samples, xenografts), from 10 ng, 50 ng and quality adjusted input amounts. MSI detection demonstrated a true positive rate of 100%.

Table 2a. SNVs and Indels: Variant detection performance using tissue DNA (FFPET)
Sample Input (ng) Total replicates Expected Variants True Positive Rate
Roche Proprietary Mix, HD753, HD789, HD200 10 8 62 100%
HD789, HD200 50 4 20 100%
HD789, HD200 Quality adjusted 4 20 100%
Table 2b. CNVs: Variant detection performance using tissue DNA (FFPET)
Sample Input (ng) Total replicates Genes Expected copies Expected Variants True Positive Rate
0710-0412


10
2 ERBB2, FGFR3 ~6 4 100%
Roche Proprietary Mix 2 ERBB2 4.7 2 100%
HD753 2 MET 4.5 2 100%
HD789 10, 50, Quality adjusted 6 (2+2+2) MET 4.5 2 100%
Table 2c. MSI: Variant detection performance using tissue DNA (FFPET)
Sample Input (ng) Total replicates Expected Status True Positive Rate
Xenografts:
- DU145
- SW48
10 4 (2+2)

MSI


100%
50 4 (2+2)
Quality adjusted 4 (2+2)
Cell-lines:
- DLD-1
- MOLT-4
- SW48
10 6 (2+2+2) MSI 100%

KAPA HyperPETE Lung Cancer Fusion Panel enables confident fusion detection from RNA samples

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

Tissue RNA (FFPET): Fusion detection and performance data

Figure 3. Performance using tissue RNA (FFPET).  High specificity was demonstrated by the high percent of reads on-target when starting from 10 ng or 50 ng of various quality (low, medium, high) input FFPE RNA. On-target rate was 92% to 97% (includes housekeeping and fusion targets) with good performance across all sample input amounts, qualities, and types. The on-target rate was calculated following rRNA read removal (0.8% to 11% of all reads). Two (2) cell-line samples (in duplicates) and fourteen (14) 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 the KAPA HyperPETE LC Fusion Panel was followed. Libraries were generated using the KAPA RNA HyperPrep kit in combination with the KAPA Universal UMI Adapters with either 10 ng or 50 ng of RNA while adjusting PCR cycles based on the input amount and 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 3.6M to 17M and data was analyzed using NAVIFY® Mutation Caller to assess enrichment and variant detection performance.

Table 3. Fusion detection performance using tissue RNA (FFPET). All fusions (100%) were detected in the reference cell line samples at both 10 ng and 50 ng RNA input amounts. Two (2) Seraseq® RNA Fusion FFPE samples and one (1) Horizon Discovery RNA Fusion FFPE sample, each run in duplicate, were used to assess fusion detection performance. The EGFR-SEPT14 variant in Seraseq® Fusion RNA Mix v4 was manually curated as the fusion caller in NAVIFY® Mutation Caller identified an EGFR partner that has a homologous sequence to SEPT14. Comparable variant detection results were achieved when down-sampling to 1M read pairs (data not shown).

Variant Type RNA Input Amount (ng) Total replicates Expected Variants True Positive Rate

Fusion

10 6 60 100%
50 6 60 100%