Quantalife

Samples were analysed using the eTAm-Seq technology in Laboratory 1 using an average of 12,450 AC per reaction. Digital PCR analysis was performed using a QX200 droplet dPCR system (Bio-Rad) with a C1000 Touch Thermal Cycler (Bio-Rad) at LGC. KRAS G12/WT and EGFR L858R/WT mutations were assessed using PrimePCR assays (Bio-Rad) and custom designed assays were used targeting NRAS A59T/WT and PI3KCA E545K/WT (S2A–S2C Table). Primers and BHQplus probes for custom assays were supplied by BioSearch and diluted in 1 x TE pH 8.0 (Sigma). Reactions (20 μL) were prepared (with 10% excess) and contained ddPCR Supermix for Probes with no dUTP (Bio-Rad), 20x primer/probe mix, 4 μL cfDNA extract (n = 1 per target mutation) with the remaining volume nuclease-free water (Ambion). Non-spiked Multiplex I cfDNA Reference Standards (32 ng/reaction) were analysed alongside the spiked extracts as controls (n = 3). Data was analysed using QuantaLife (Bio-Rad, version 1.6.6.0320) with classification of single positive, double positive and negative droplets as shown in S1A–S1E Fig. Copy number concentration was calculated based on a partition volume of 0.85 nL.

For the quantification of tumor tissue RNA, real-time qPCR was performed on 7500 Real-Time PCR systems (Applied Biosystems, Waltham, USA) using TB Green Fast qPCR Mix from TaKaRa (Cat: RR430A, Beijing, China). The relative level of targets was analyzed by the ΔΔCt method using GAPDH mRNA as the internal control. In the case of serum RNA, droplet digital PCR was carried out for absolute quantification using droplet digital polymerase chain reaction (ddPCR) Supermix on a BioRad QX200 Droplet Digital PCR System (Munich, Germany) following the manufacturer's instruction. Briefly, 20 μl PCR was mixed with 70 μl droplet generator oil pipetted into the cavities of the droplet generator cartridges (Bio-Rad) to generate droplet, and then, 40 μl droplet suspension was transferred into 200 μl PCR tubes. After a brief spin down, the tubes were located into the PCR machine to perform the program of 95°C for 10 min followed by 41 × 95°C for 30 s and 60°C for 60 s and 98°C for 10 min. The level of each target was detected using a QX200 droplet reader, and the data analysis was analyzed using the QUANTALIFE (Bio-Rad) software. All the involved primers for PCR were listed in Table 2.