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.
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%.
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% |
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% |
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% |
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% |