Plasma Isolation and Cell-free DNA Extraction

Blood was collected into acid-citrate dextrose (ACD) blood tubes and processed within four hours of collection using standard blood processing methods. Briefly, whole blood was centrifuged once at 1300 × g for 10 min in a swinging bucket centrifuge with the brakes turned off. The upper plasma was collected without disrupting the buffy coat layer, aliquoted (1 mL/aliquot) into prelabeled cryovials, and stored frozen at −80 °C. Cell-free DNA was isolated from 2 mL of ACD plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen) following the manufacturer’s protocol for the vacuum manifold method and then sequenced.