Fig. 2

a Comparative ddPCR analysis of a dilution series of the indicated synthetic ESR1 E380Q oligonucleotide. We used serial dilutions of the ESR1 E380Q molecule and analyzed them by ddPCR. A mixture of recombinant ESR1 E380Q and WT was plotted against the different fractional concentrations from 32 to 0 copies/μL. The MAF of ESR1 E380Q was maintained at more than two droplets. Therefore, a mutation was only considered to be present if more than two positive droplets were detected. Abbreviations; ddPCR, droplet digital polymerase chain reaction; WT, wild-type; MAF, mutant allele frequency. b-d Dilution experiments where ESR1 E380Q oligonucleotide was diluted in a background of WT normal human DNA are shown. The dilution experiments were prepared by 1.3-fold serial dilution of synthetic ESR1 E380Q stock oligonucleotide in a background of WT normal human DNA (TaqMan Control Genomic DNA) where the total DNA content of each ddPCR reaction was 20 ng and “wild-type double” was 40 ng. b The box plots of ESR1 E380Q and ESR1 WT detected in each input DNA. c, d The fluorescent signal (C: ESR1 E380Q, d: ESR1 WT) for each droplet is plotted on the y-axis for each dilution, which is separated by a dotted yellow line, with input DNA indicated b. The positive droplet fluorescence threshold is indicated by the magenta line. Blue dots represent FAM-labeled ESR1 E380Q mutant DNA (C), green dots represent HEX-labeled WT DNA (d), and black dots are droplets with no DNA incorporated. Each droplet is cumulatively counted as an ‘Event Number’ for the ddPCR experiments analyzed in tandem, and plotted along the x-axis. Abbreviations; WT, wild-type; ddPCR, droplet digital polymerase chain reaction