D1000 reagents

Low-pass sequencing of 42 formalin-fixed paraffin-embedded (FFPE) samples of melanoma was performed by Psomagen (Rockville, MD, USA). Briefly, the input DNA quality was verified using gel electrophoresis, and the quantity was measured using the Picogreen assay (Thermo Scientific, Waltham, MA, USA); 2 ng of DNA was prepared in 30 ul of buffer and used for library construction using the Nextera DNA Flex Library Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s guidelines. The size of the final DNA libraries was then validated using the TapeStation D1000 ScreenTape (Agilent, Santa Clara, CA, USA) and D1000 reagents (Agilent, Santa Clara, CA, USA). The quantity was measured using the Picogreen assay (Thermo Scientific, Waltham, MA, USA), and the molar concentration was calculated using both sources. The libraries were normalized to 2 nM, pooled in equimolar volume, and then loaded on the flow cell from the Novaseq S4 300 cycle kit (Illumina, San Diego, CA, USA) and the XP-4lane kit (Illumina, San Diego, CA, USA). The prepared flow cell and SBS cartridge from the Novaseq S4 300 cycle kit (Illumina, San Diego, CA, USA) were inserted into the Novaseq 6000 system and sequenced using 151-10-10-151 running parameters, reaching a mean depth of 0.48 X.

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 10⁴AKT1 copies per mL plasma, taking into consideration the low presence of wild-type AKT1 in the plasma (S1 Table).