Qx200 digital reader

DNA was extracted from screening or pre-treatment C1D1 plasma (to match the timepoint sequenced by targeted sequencing with Guardant360) using the automated QiaSymphony platform (Qiagen, Hilden) or manually using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden) following the manufacturer’s instructions. Extracted DNA was quantified by Qubit (Thermo Scientific) or by Taqman assay droplet digital PCR (ddPCR). Following quantification, DNA was combined with singleplex ddPCR assays targeting specific mutations (Table 1 and Supplementary Table 4), supermix and nuclease free water before partition into droplets using an Automated Droplet Generator (Bio-Rad, Pleasanton). Samples underwent 40 cycles of PCR on a thermal cycler before analysis on a QX200 Digital Reader (Bio-Rad, Pleasanton). ddPCR assays were considered positive with one or more positive FAM droplets (mutant) per reaction. Poisson probability was used to calculate allele frequency (AF).Table 1

PIK3CA and HER2 (ERBB2) mutations identified within Guardant Health sequencing validated by droplet digital PCR (ddPCR).

Gene	Mutation
PIK3CA	E545K
PIK3CA	E542Q
PIK3CA	E726K
PIK3CA	E545Q
PIK3CA	H1047R
HER2	G727A
HER2	E717D
HER2	Q711H
HER2	L786V
HER2	I628M
HER2	D1105N
HER2	S1002N
HER2	L800R
HER2	L800P
HER2	Q1206K
HER2	R1153Q
HER2	E1079K
HER2	V777M
HER2	D769H
HER2	D769Y
HER2	L755S
HER2	I767M
HER2	S310F

Following enrolment into plasmaMATCH, patients had a blood sample drawn which underwent prospective ctDNA testing for hotspot mutations in PIK3CA, ESR1, HER2 and AKT1 (Supplementary Fig. 1b and Supplementary Table 4, and methodology previously published⁴⁴ ). DNA was extracted from screening plasma using the automated QiaSymphony platform (Qiagen, Hilden). DNA was quantified by Qubit (Thermo Scientific) before being combined with singleplex or multiplex droplet digital PCR (ddPCR) assays targeting hotspot mutations⁴⁴ (Supplementary Table 4), supermix and nuclease free water. The samples were partitioned into droplets using an Automated Droplet Generator (Bio-Rad, Pleasanton) before undergoing 40 cycles of PCR on a thermal cycler. Results were analysed on a QX200 Digital Reader (Bio-Rad, Pleasanton). ddPCR assays were considered positive with a minimum of two positive FAM droplets (mutant) per reaction. Poisson probability was used to calculate allele frequency (AF).