Supermix for probes

Primers and probes (listed in Supplementary Table 3) were diluted to concentrations of 900 nM and 250 nM per primer and probe, respectively. Overall, 22 µL of reactions were prepared with 11 µL of Bio-Rad SuperMix for Probes (1× final concentration), 6.6 µL of diluted primers (900 nM/primer, final concentration) and probes (250 nM/probe, final concentration), and 4.4 µL of diluted cDNA. In all, 20 µL of each reaction mixture was partitioned into droplets using a Bio-Rad QX200 droplet generator per the manufacturer's instructions. PCR conditions were: (1) 95 °C for 10 min, (2) 40 cycles of A) 94 °C for 30 s and B) 57 °C for 1 min, (3) 98 °C for 10 min, and hold at 4 °C. Droplets were then read on Bio-Rad QX200 droplet reader, and the number of cDNA copies/µL was calculated.

Digital droplet PCR (ddPCR) was used to quantify DNA in affinity purified and total DNA samples. Typically, samples were diluted 100 to 10000 to bring the target DNA concentration within ddPCR dynamic range. PCR reactions (25 μl) contained supermix for probes (BioRad), 0.5 mM of forward and reverse primers, 0.25 mM TaqMan probe and 1μl of the diluted sample. PCR reactions were processed in an automatic droplet generator (BioRad) before performing the PCR cycles. Droplets were analyzed in a QX200 droplet reader using QuantaSoft software package (BioRad). GFP DNA was quantified using primers and probe DB307, DB308 and DB309. URA3 was quantified using primers and probe DB406, DB407 and DB408. 18S rDNA was quantified using primers and probe DB438, DB439 and DB440. GFP and URA3 copy number in samples from total yeast DNA were reported relative to the 18S rDNA copy number in each sample for normalization.

Supermix for probes, Droplet digital PCR (ddPCR) assays, DG8TM cartridges and Droplet Generation Oil were obtained from Bio-Rad Laboratories Ltd., Hertfordshire, UK. Deionized water, sodium chloride, sodium hydroxide, phosphate-buffered saline (PBS), sodium nitrate, 4-aminobenzoic acid, hydrochloric acid, ethanolamine, 2-(N-morpholino) ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hexammineruthenium (III) chloride, potassium ferricyanide, potassium chloride and potassium ferrocyanide were all purchased from Sigma–Aldrich, (Dorset, UK). Two hundred and fifty units of HotStarTaq Plus and dNTP Mix, PCR Grade (200 μL), were purchased from Qiagen, (Manchester, UK). Phusion Direct PCR kit was purchased from thermo fisher scientific (Renfrew, UK).

The ddPCR platform (Qx200 ddPCR system, Bio-Rad) was used for the detection of KRAS mutations in cfDNA as per the manufacturer's instruction. Twenty microliter ddPCR reaction solutions were prepared with dUTP-free Supermix for probes (Bio-Rad), 900 nM of primers, 250 nM of hydrolysis probes and at least 0.5 ng of cfDNA. The amplification was performed under the following conditions: 95°C for 10 min, 40 cycles of (94°C for 30 s, 60°C for 60 s), 98°C for 10 min. The results were analyzed by the Quanta-Soft Analysis Pro software (Bio-Rad).

cDNA was synthesised as described for RT-qPCR. No pre-amp was performed. Undiluted cDNA, 3 µL, was mixed with 10.0 µL Supermix for Probes (P/N 186-024, Bio-Rad), 1.0 µL TaqMan microRNA Assays (20 ×) and 6.0 µL nuclease-free water. The 96-well plate was sealed and droplets generated by the Automated Droplet Generator and Droplet Generation Oil for Probes (P/N 1864110, Bio-Rad). The cycling condition was 15 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, then 98 °C for 10 min and cooling to 4 °C. Ramp rate was 2 °C/s. A gradient of annealing temperature 56–62 °C was tested. The separation of positive and negative droplets was good at 60 °C, which is the recommended annealing temperature for both Supermix for Probes and the Small RNA Assays (Supplementary Information and Data 1). Droplets were analysed using the QX200 Droplet Reader and the QuantaSoft Analysis Pro software (Bio-Rad). The thresholds for positive droplets were amplitude 3500 for miR371 and 4000 for miR30b across all samples. Triplicate wells were analysed for all samples.
The miR371 and miR30b concentrations from RT-ddPCR are presented as copies/µL serum if not otherwise stated and were calculated from the instrument output given in copies/µL PCR reaction (see Supplementary Information and Data 2).

A duplex ddPCR assay was adapted using separate indicators for PfMGET and Pfs25 markers in a single reaction, and Pfs16 was quantified in a separate reaction (Pomari et al., 2020 (link)). For both assays, each reaction included 11 μL Supermix for Probes (no dUTPs) (Bio-Rad Laboratories Inc (2018) ., Hercules, CA, US), 2 μL cDNA template, nuclease free water as needed, 455 nM of each primer and 250 nM of each probe in a final reaction volume of 22 μL. Droplets were generated using the QX200 Droplet Generator (Bio-Rad Laboratories Inc (2018) , Hercules, CA, US) within a range of 10,000 to 20,000 droplets. Reactions were performed on the C1000 Touch Thermal Cycler (Bio-Rad Laboratories Inc (2018) , Hercules, CA, US) under the following programs (ramp rate 2°C/second): Duplex PfMGET/Pfs25 - 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds and 50°C for 1 minute, 98°C for 10 minutes; Pfs16 - 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds and 55°C for 1 minute, 98°C for 10 minutes. Droplets were analyzed and counted on a QX200 Droplet Reader (Bio-Rad Laboratories Inc (2018) , Hercules, CA, US).

For each single-target gene ddPCR assay, the reaction mixture (20 μL) contained 10 μL Supermix for probes (Bio-Rad, USA), 900 nM forward primer, 900 nM reverse primer, 100–1500 nM TaqMan probe, 2 μL DNA template, and ddH₂O.
For the multiplex ddPCR assay, the reaction mixture (20 μL) contained 10 μL Supermix for probes (No dUTP) (Bio-Rad), 900 nM each forward primer (ypo2088-F, caf1-F, and pla-F), 900 nM each reverse primer (ypo2088-R, caf1-R, and pla-R), 250 nM pla-T, 250 nM caf1-T, 500 nM ypo2088-T, 2 μL DNA template, and ddH₂O.
Next, 20 μL ddPCR reaction mixture and 70 μL Droplet generation Oil For Probe (Bio-Rad) were used to generate droplets with the QX200 ddPCR system (Bio-Rad). The droplets were amplified using a T100 Thermal Cycler (Bio-Rad) under the following conditions: initial denaturation at 95°C for 10 min, 40 cycles of denaturation (94°C, 0.5 min), and annealing/extension (60°C, 1 min), enzyme deactivation (98°C, 10 min), and incubation at 12°C for 30 min. After amplification, the products were detected and analyzed with a droplet reader in the QX200 ddPCR system.