AKT1 E17K ctDNA reference standards

The ctDNA reference standard was developed by Horizon Discovery (Cambridge, UK) using an AKT1 E17K heterozygous cell line and corresponding AKT1 wild-type cell line (catalogue numbers HD658 and HD659). Genomic DNA was extracted from both cell lines using the Maxwell 16 DNA purification Kit (Promega, Madison, WI) according to manufacturer’s instructions. Extracted DNA was quantified using a NanoDrop spectrophotometer, diluted to 50 ng/μl, and sheared to an average peak size of 170 bp using the Covaris sonicator (Covaris, Woburn, MA). Fragment size was measured by the Agilent TapeStation instrument using D1000 reagents (Agilent Technologies, Santa Clara, CA). Sheared DNA was quantified using Qubit® dsDNA BR Assay kit (Thermo Fisher Scientific), according to manufacturer’s instructions. Sheared DNA of both cell lines was blended to obtain 0.05%, 0.5%, 1%, 2% and 5% mutant allele frequencies, assuming 3.3 pg per genomic equivalent. The admixtures were spiked into pooled healthy donor plasma (described above) to obtain 6 x 10⁴ AKT1 copies per mL plasma, taking into consideration the low presence of wild-type AKT1 in the plasma (S1 Table).