Using the Qiagen Therascreen EGFR, KRAS, and BRAF RGQ kits as reference methods in this study, we were able to validate detected hotspot variants down to 4% VAF from the NGS analysis in both the validation cohort and the subsequent prospective cohort in all tested cases, thus representing the effectively used limit of detection in later clinical samples (notably, a strict 10% tumor percentage cut-off was used for decision to perform clinical mutation testing at all). This evidence concerns the gene EGFR and neoplasm.