Pierceable sealing foil sheets

Each sample was performed in two replicates. For each replicate, 5 μL of DNA was digested with the restriction enzyme HindIII in NEB Buffer 2 (New England Biolabs, Ipswich, MA, USA) for 30 min at 37°C, and then diluted 1:2.5 with molecular biology grade water. A 20 μL of mixture of primers and probes, Bio‐Rad 2X Supermix, and the digested diluted DNA was emulsified with droplet generator oil (Bio‐Rad, Hercules, CA, USA) using a QX‐100 droplet generator according to the manufacturer's instructions. The droplets were then transferred to a 96 well reaction plate (Eppendorf, Hauppauge, NY, USA) and heat‐sealed with pierceable sealing foil sheets (Thermo Fisher Scientific, West Palm Beach, FL, USA). PCR amplification was performed in this sealed 96 well plate using a GeneAmp 9700 thermocycler (Applied Biosystems, Grand Island, NY, USA) with the following cycling parameters: 10 min at 95°C, 40 cycles consisting of a 30‐s denaturation at 94°C and a 60‐s extension at 59°C, followed by 10 min at 98°C and a hold at 12°C. Immediately following PCR amplification, droplets were analyzed using a QX100 droplet reader (Bio‐Rad), in which droplets from each well are aspirated, streamed toward a detector and aligned for single‐file two‐color detection (Hindson et al, 2011, Analytical Chemistry).⁴⁹