Validation of selective KRAS and BRAF mutations detected by qBiomarker with ultra-sensitive ddPCR.(A) List of cancer-associated KRAS and BRAF mutation loci assessed by qBiomarker and validated with ddPCR assay. (B) Serial dilution curve using DNA with known KRAS mutation (extracted from the PDX tumour CTG-0288). The blue markers indicate the concentration of mutant DNA (copies per μl) and the orange markers indicate the fractional abundance (%) of the KRAS mutated loci in a wild-type DNA background. All error bars generated by QuantaSoft software (Bio-Rad, Hercules, CA, USA) represent a 95% confidence interval. (C) To assess cross-reactivity of ddPCR probes targeting KRAS p.G12D and p.G12V mutations, DNA with known KRAS p.G12D mutation was probed with either specific ddPCR assay or with a probe designed for detection of p.G12V substitution. Alternatively, DNA isolated from tumour carrying the KRAS p.G12V mutation (D) was probed with either a specific or off-target ddPCR probe. Blue dot clusters indicate KRAS mutation detected by the specific assay. Black dot cluster indicates empty droplets. Mutated KRAS p.G12V cases when probed with the assay for p.G12D, and vice-versa, presented with an extra shifted cluster of black dots (identified by a star), probably resulted due to non-specific probes cross-reactivity. Green clusters indicate droplets containing wild-type KRAS alleles. (E) Schematic representation of the comparative analysis of qBiomarker and ddPCR approaches in a panel of 104 PDX tumours. Green and blue dots represent mutations discovered by either qBiomarker or ddPCR, respectively, although red dots indicate mutations concurrently detected by both platforms. Cases with no mutation detected are not shown. (F) Venn diagram summarising the number of KRAS and BRAF cancer-associated mutations detected by qBiomarker and ddPCR alone or concurrently. (G) Summary of detection accuracy (specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of the qBiomarker approach when referenced to ddPCR.