Circulating cfDNA was isolated from 2 to 4 mL of plasma using the Qiagen QIAamp Circulating Nucleic Acids Kit (Qiagen GmbH), eluted in 52 μL of RNase-free water containing 0.04% sodium azide (Qiagen GmbH), and stored in LoBind tubes (Eppendorf AG) at −20°C. Concentration and quality of cfDNA were assessed using the Bioanalyzer 2100 (Agilent Technologies).
Next-generation sequencing cfDNA libraries were prepared for whole-genome sequencing using 15 ng cfDNA when available or entire purified amount when less than 15 ng (Supplementary Table S5). In brief, genomic libraries were prepared using the NEBNext DNA Library Prep Kit for Illumina (New England Biolab) with four main modifications to the manufacturer's guidelines: (i) the library purification steps followed the on-bead AMPure XP (Beckman Coulter) approach to minimize sample loss during elution and tube transfer steps; (ii) NEBNext End Repair, A-tailing, and adapter ligation enzyme and buffer volumes were adjusted as appropriate to accommodate on-bead AMPure XP purification; (iii) Illumina dual index adapters were used in the ligation reaction; and (iv) cfDNA libraries were amplified with Phusion Hot Start Polymerase. All samples underwent a 4-cycle PCR amplification after the DNA ligation step.
Next-generation sequencing cfDNA libraries were prepared for whole-genome sequencing using 15 ng cfDNA when available or entire purified amount when less than 15 ng (Supplementary Table S5). In brief, genomic libraries were prepared using the NEBNext DNA Library Prep Kit for Illumina (New England Biolab) with four main modifications to the manufacturer's guidelines: (i) the library purification steps followed the on-bead AMPure XP (Beckman Coulter) approach to minimize sample loss during elution and tube transfer steps; (ii) NEBNext End Repair, A-tailing, and adapter ligation enzyme and buffer volumes were adjusted as appropriate to accommodate on-bead AMPure XP purification; (iii) Illumina dual index adapters were used in the ligation reaction; and (iv) cfDNA libraries were amplified with Phusion Hot Start Polymerase. All samples underwent a 4-cycle PCR amplification after the DNA ligation step.