2.4 | Droplet digital PCR assayFor

2.4 | Droplet digital PCR assayFor mutation detection, we used the ddPCR platform QX100 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, CA, USA), in- cluding primers and probes (FAM, mutant type; HEX, wild type), and ddPCR Supermix for Probes (No dUTP) according to the manufactur- er's protocol. For every experiment, we used a positive control cor- responding to each mutation. Primers and probes for ddPCR were TERT promoters C228T/C250T, FGFR3 S249C, and PIK3CA E545K(Table S1). Droplets were generated using a droplet generator (Bio- Rad Laboratories). The PCR cycle for FGFR3 included a 10-minute incubation at 95°C followed by 40 cycles at 94°C for 30 seconds and at 55°C for 1 minute, one cycle at 98°C for 10 minutes, and a 12°C hold; and that for the TERT promoter included a 10-minute incubation at 95°C followed by 50 cycles at 94°C for 30 seconds and at 55°C for 1 minute, one cycle at 98°C for 10 minutes, and a 12°C hold. We used 5.7 ng (range: 1.5-30.7 ng) of urinary cfDNA for each ddPCR analysis. Droplet fluorescence was assessed in a droplet reader. Analysis of ddPCR data for allele calling and calculation of ab- solute copy numbers were done using QuantaSoft software version1.7.4 (Bio-Rad Laboratories). The samples were considered positive for targeted mutations when they met two criteria: (i) they contained at least three droplets in the positive area of the FAM signal; and (ii) the MAF was >0.1%. MAF was defined as the proportion of copies of the mutant type relative to the sum of copies of the mutant and wild type obtained by the ddPCR platform.