Phusion high fidelity 2x master mix

Per sample, 2 to 10 ng of cfDNA was used for PCR-based enrichment of TP53 sequences.
Briefly, 20 μl reaction mixture composed by 10 μl of Phusion High-Fidelity Master Mix 2X (NEB), 2 μl of Betaine 5M (Sigma-Aldrich), 0.5 μl of pure DMSO (Sigma-Aldrich), 0.5 µM of the forward and the reverse primers each, cfDNA, and MilliQ (to a volume of 20 μl)) was prepared. If necessary, the volume of the cfDNA was reduced by SpeedVac at medium temperature prior to preparation of the PCR mix. The PCR reaction consisted of 1 minute incubation at 98°C, 30 cycles (Supplementary Table ) of 30 seconds at 98°C and 15 seconds at 59°C, and finally 2 minutes incubation at 72°C. PCR products were gel-purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's protocol.
PCR products were kept at -20°C. Sample CY_SM_PC_HC_0004_003 and CY_SM_PC_HC_0004_004 were amplified using the CleanPlex TP53 Panel of Paragon Genomics according to manufacturer's protocol.

The backbones were generated by annealing and fill-in of two semi-complementary synthetic phosphorylated oligos purchased from Integrated DNA Technologies (https://www.idtdna.com).
A polymerase with error-correction activity was used for the fill-in reaction in order to obtain blunt-end products, with phosphorylated ends. The reaction consisted of a 25 µl Phusion High-Fidelity Master Mix 2X (New England Biolabs), 23 µl of water and 1ul of each oligo at a concentration of 10 µM. The reaction was subjected to 5 cycles of DNA melting (1 minute at 98°C), annealing (30 seconds at 65°C) and elongation (15 seconds at 72°C). All the backbones were gel-purified.