Fig. 1

Design of the dPCR assay using LNA-hydrolysis probes for detecting the EGFR amplification and EGFRvIII variant. a Three amplicons were designed within the EGFR gene from Universal Probe Library® (Sigma-Aldrich). EGFR1, EGFR2 and EGFR3 are located within three different regions in the gene. EGFR2 is inserted into the deleted region of the EGFRvIII variant (deletion of exons 2–7). b Two-dimensional cluster plot representing the 6-carboxyfluorescein (FAM)-labeled LNA-based hydrolysis probe for the EGFR-targeted sequence (EGFR1, EGFR2 or EGFR3) against the trichloro-phenylcarboxyfluorescein oligonucleotide (VIC)-labeled hydrolysis probe for the HMBS amplicon. Droplets are grouped as clusters: FAM/VIC-negative (double-negative droplets, blue), FAM-positive/VIC-negative (green), FAM-negative/VIC-positive (pink), and FAM/VIC-positive (double-positive droplets, orange). The EGFR copy number was determined by calculating the ratio of EGFR FAM-labeled droplets over the HMBS VIC-labeled droplets multiplied by the number of HMBS copies (× 2 in the human genome)