Qx100 system

The ddPCR was performed using the QX100 system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations. A 20 µL ddPCR reaction mixture contained 1 × ddPCR master mix (Bio-Rad, Hercules, CA, USA), 0.9 µM primers, 0.25 µM probe (Table 1) and 2 mL of tested cDNA. The entire reaction mixture and 70 µL of droplet generation oil (Bio-Rad, Hercules, CA, USA) were loaded into a disposable plastic cartridge (Bio-Rad, Hercules, CA, USA) and placed in a droplet generator. After processing, the droplets obtained from each sample were transferred to a 96-well PCR plate. The amplification was carried out using T100TM Thermal Cycler (Bio-Rad, Hercules, CA, USA) according to the program: DNA polymerase activation at 95 °C for 10 min followed by 45 cycles of PCR amplification (94 °C for 30 s and 58 °C for 60 s), and 98 °C for 10 min, 2 °C/s⁻¹ ramp rate at all steps. After PCR, the droplets were counted using a QX100 Droplet Reader. The data obtained were analyzed using QuantaSoft Analysis Pro^TM 1.0.596 software (Bio-Rad, Hercules, CA, USA).

ddPCR was performed using the Bio-Rad QX100 system (Bio-Rad, Hercules, CA, USA) and QuantaSoft for data analysis. There are two sets of primers and probes targeting the VP1 and large T antigen regions, respectively (Table 6). Each reaction was performed with Bio-Rad ddPCR supermix for probes with the final concentration of primers at 900 nM and probes at 250 nM and 25 units of HindIII (New England Biolabs). Plasmid BK Dunlop and JC Mad-1 were gifts from Peter Howley (Addgene plasmids no. 25466 and no. 25626) and were used as positive controls for 1:1 VP1/large T-antigen copy number. After droplet generation, droplets were transferred to a 96-well PCR plate and amplified on a 2720 thermal cycler (Applied Biosystems) with the following thermocycling parameters: 94°C for 10 min, followed by 40 cycles of 94°C for 30 s and 60°C for 1 min, and 98°C hold for 10 min. After the thermal cycling, the plate was transferred to a droplet reader. The QuantaSoft software was used for data analysis.

Optimized TaqMan MGB probe PCR assays targeting the mutation site for wild type and 7 mutant types were conducted as described in Table S1 in the supplemental material. The ddPCR analysis was performed on a QX100 system (BioRad Laboratories, Inc., Shanghai, China). The reaction mixture was in a volume of 20 µL and comprised 10 µL of 2× ddPCR Super Mix for Probe (BioRad Laboratories, Inc., Shanghai, China), 1 µL of 5 µM primers mixture, 0.2 µL of 5 µM wild type probe labeled with VIC (Thermo Scientific, Beijing, China), 0.2 µL of 5 µM mutant probe labeled with FAM (Thermo Scientific, Beijing, China), 6.6 µL of ddH₂O and 2 µL of template DNA with a concentration of 25 ng/µL.
The optimized PCR thermal profile was conducted on a conventional PCR machine (Vetiti, Applied Biosystems). Thermal cycling consisted of a 10 min activation period at 95 °C followed by 40 cycles of a two-step thermal profile of 15 s at 95 °C denaturation and 60 s at 60 °C for combined annealing-extension and 1 cycle of 98 °C for 10 min. All samples were analyzed in three replicates. Results were analyzed with the QuantaSoft v.1.2.10.0 software (BioRad Laboratories, Inc., Shanghai, China). The workflow and data analysis were described in our previous report^{25 (link)}.