Fig 1.
Flow chart for variant analysis.
This chart is used for our classification of variants in solid tumor samples. VAF: Variant allele frequency; CDS: coding sequence; SNP: single nucleotide polymorphism; MAF: Minor allele frequency; IGV: Integrative Genomics Viewer; LoF: Loss-of-function.
Fig 2.
Correlation of dCt with mean coverage.
Plot showing the correlation of the DNA quality (QC), expressed as dCt, with the mean coverage (log scale) of 26 random FFPE validation samples.
Table 1.
The accuracy of the NGS workflow was checked with the Horizon Quantitation multiplex reference that contains 11 mutations at known variant allele frequencies (VAF).
NGS data are provided for DNA start amounts of 100 ng and 200 ng. Mean and standard deviations (SD) are given as well. del: deletion; nr: not reported.
Fig 3.
Two samples with known mutations were mixed at different percentages, captured and sequenced. VAFs of each mutation were plotted against the percentage in the mix. (A) Mutations KRAS G13D (58%), FBXW7 R505S 48%), APC K1462fs (52%), NRAS Q61R (35%) with high VAFs in the original sample. (B) Mutations NRAS Q61L (9%), ERBB2 V842I (8%), TP53 Y234* (9%), KRAS D12D (24%) with low VAFs in the original sample.
Table 2.
Targeted NGS analysis on 150 diagnostic FFPE samples.
Number (#) and percentages (%) of samples and variants are indicated for the three tumor types investigated. Only pathogenic or presumed pathogenic variants that met the acceptance criteria (Cov >300; VAF >5%) were included. Many samples had more than one mutation. Insuff reads: mean coverage <300. total #: total number of variants.
Table 3.
Summary of the validation assays and results for the targeted NGS screening on solid tumors (NSCLC, CRC and MELA).