Sample preparation, 5hmC-Seal, and sequencing

Details about cfDNA sample preparation, 5hmC-Seal library construction, and subsequent sequencing have been described in our previous publications^14,15. Briefly, ~2–3 mL of frozen plasma from each subject was processed by centrifuging at 1350 × g for 12 min twice and at 13,500 × g for 12 min once, followed by cfDNA extraction (~2–4 ng/sample) using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). The 5hmC-Seal libraries were constructed according to an established protocol^14,36. DNA samples were first repaired and ligated with adaptors. Next, T4 bacteriophage enzyme β-glucosyltransferase was used to transfer an engineered glucose moiety containing an azide-glucose to 5hmC in duplex DNA. A biotin tag was added to the azide group using Huisgen cycloaddition (“Click”) chemistry. Finally, the 5hmC-containing DNA fragments with biotin tags were captured by streptavidin beads. The 5hmC-Seal libraries were constructed through PCR amplification and paired-end sequenced using the Illumina NextSeq 500 platform (PE50) at the University of Chicago Genomics Core Facility. The cfDNA samples were randomly labeled for the 5hmC-Seal library construction and sequencing. Technicians did not have access to clinical outcomes. Technical robustness, including reproducibility and spike-in controls, of the 5hmC-Seal have been demonstrated in our previous studies^14,36,37.