Cell-free DNA was extracted using the QIAmp Circulating cfDNA Extraction Kit (Qiagen) 3-8 ng cfDNA was used in the 5hmC-Seal assay (see Li W, Zhang X, Lu X, You L, Song Y, Luo Z, et al. 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res 2017;27:1243-57). Libraries were constructed on-bead using the KAPA HyperPlus kit (Roche).