For each study subject, approximately 2-3 mL of frozen plasma was processed by centrifuging twice at 1,350 × g for 12 min, and then centrifuging at 13,500 × g for 12 min, followed by cfDNA extraction using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). Each cfDNA sample was first repaired and ligated with adaptors. Next, the T4 bacteriophage enzyme β-glucosyltransferase (β-GT) was used to transfer an engineered glucose moiety containing an azide-group to 5hmC in duplex DNA. A biotin tag was then installed onto the azide group using Huisgen cycloaddition (“click”) chemistry, followed by capturing of 5hmC-containing DNA fragments using avidin beads. The 5hmC-Seal library for each cfDNA sample was then constructed through PCR amplification, followed by paired-end sequencing (PE38) on the Illumina NextSeq500 platform at Shanghai Epican Genetech Co. Ltd. (Shanghai, China), according to the manufacturer's protocol. We randomized cfDNA samples when constructing the 5hmC-Seal libraries and sequencing. The 5hmC-Seal was shown to be technically robust and stable in our previous studies using duplicate samples and spike-in controls. 5hmC-Seal
GEO Accession
GSM4697700