PCR droplets were generated on the RDT1000 (RainDance Technologies, 20-01000) using the manufacturer's recommended protocol: 25 μl of the DNA template mixture in a tube, the custom primer droplet library in a separate tube and a disposable microfluidic chip (RainDance Technologies) were placed onto the RDT1000. The custom primer droplet library consisted of the primer pairs described in the 'Library primer design' section above where each primer droplet contained matched forward and reverse PCR primer pairs (5.2 μM per primer). The RDT1000 generates each PCR droplet by merging a single DNA template droplet with a single primer droplet [13]. Each PCR droplet contains a final primer concentration of 1.6 μM per primer. The PCR droplets are automatically dispensed as an emulsion into a PCR tube per each test sample and transferred to a standard thermal cycler for PCR amplification. Each single sample generated more than 1,000,000 single plex PCR droplets.
