1. Total and mutated EGFR quantification in cell-free DNA from non-small cell lung cancer patients detects tumor heterogeneity and presents prognostic value Alegre E, Fusco JP, Restituto P, Salas-Benito D, Rodríguez-Ruiz ME, Andueza MP, Pajares MJ, Patiño-García A, Pio R, Lozano MD, Gúrpide A, Lopez-Picazo JM, Gil-Bazo I, Perez-Gracia JL, Gonzalez A Tumor Biology Corresponding author: Alvaro Gonzalez. Clinica Universidad de Navarra. agonzaleh@unav.es

2. Supplementary Fig. 1: Schematic representation of patients and cfDNA samples included in the study Basal N=18 Best response N=13 Pre-progression N=7 Progression N=12 EGFR-TKI treatment 36 NSCLC patients with EGFR mutations detected in tissue biopsy 7 early stage NSCLC patients 29 advanced stage NSCLC patients 24 patients treated with EGFR-TKI 5 patients not treated with EGFR-TKI 5 basal samples 7 basal samples

3. Wild-type DNA for L858R mutation Positive cfDNA sample for L858R mutation Wild-type / HEX amplitude Mutant / FAM ampllitude Supplementary Fig. 2: 2-D Fluorescent amplitude plots for L858R (A), delE746-A750 (B) and T790M mutations corresponding to DNA from PBMC from a healthy control (above) and cfDNA from a NSCLC patient positive for the corresponding mutation (bottom). Black cluster on the plot corresponds to negative droplets, green cluster corresponds to droplets positive for wild type, blue cluster corresponds to droplets positive for the corresponding mutation, and orange cluster corresponds to droplets positive for both wild type and the corresponding mutation. Mutant / FAM ampllitude Wild-type / HEX amplitude Wild-type DNA for delE746-A750 mutation Positive cfDNA sample for delE746-A750 mutation Wild-type DNA for T790M mutation Positive cfDNA sample for T790M mutation Wild-type / HEX amplitude ABC

4. Supplementary Fig. 3: Correlation between EGFR mutated copies for L858R and delE746-A750 mutations and mutated copies for T790M mutation, in cfDNA from samples positive for EGFR mutations.