GSM9229563: sample 34 Control; Homo sapiens; Bisulfite-Seq
cfDNA was extracted and prepared for sequencing using a harmonized protocol at UCSF and UQ. Plasma (2–8 ml) was thawed at room temperature, and cfDNA was isolated with the QIAGEN Circulating Nucleic Acid kit (Cat. No. 55114). DNA was quantified with the Qubit dsDNA HS assay and assessed using the Agilent TapeStation 4200 (UCSF) or Bioanalyzer 2100 HS kit (UQ). Bisulfite conversion was performed with the Zymo Lightning kit, and libraries were prepared using the Accel-NGS Methyl-Seq kit (Swift Biosciences) with one major modification: denatured, bisulfite-converted cfDNA underwent adaptase, extension, and ligation, followed by ligation cleanup and primer extension (98 °C 1 min; 70 °C 2 min; 65 °C 5 min; 4 °C hold) with UMIs and i5 barcodes to enable duplicate removal and accurate methylation estimation.After exonuclease I treatment and purification, libraries were amplified with a universal P5 primer and custom i7-barcoded P7 primers (98 °C 30 s; 15 cycles of 98 °C 10 s, 60 °C 30 s, 68 °C 60 s; final 68 °C 5 min; 4 °C hold). Unique-dual indexed libraries were purified, quantified (Qubit HS-dsDNA), quality-checked (Agilent D1000-HS assay), and pooled in 12-plex. Pools underwent hybridization capture (xGen Hybridization Capture Kit, IDT) using custom probes targeting ~5,000 regions. Captured libraries were re-amplified (98 °C 30 s; 10 cycles of 98 °C 10 s, 60 °C 30 s, 68 °C 60 s; final 68 °C 5 min; 4 °C hold), purified with SPRI beads, and quantified. Identical probes were used across sites for consistency. Final pools were sequenced on an Illumina NovaSeq 6000 (S4 lane, 150 bp PE, 8 bases i7, 17 bases i5) at UCLA (USA) and UNSW Ramaciotti (Australia).