Fig. 1

Overview of library construction for traditional enzymatic methyl-seq and NEEM-seq. A The process of traditional enzymatic methyl-seq to detect methylated (5mC) and hydroxymethylated (5hmC) cytosines. Firstly, the DNA fragments underwent end-repair and dA-tailing processes, followed by P5 and P7 adapter ligation. Artificially unmethylated cytosines located in CpG sites (indicated by red dashed boxes) were introduced to the cfDNA in end repair. The enzymes TET2 and T4-BGT, oxidized 5mC and 5hmC to 5-carboxycytosine and 5-(β-glucosyloxymethyl) cytosine (5caC/5gmC) to prevent from deamination in the subsequent step. Next, unprotected cytosines were converted to uracils by the deaminase of APOBEC and amplified by PCR for sequencing. B The procedures of our NEEM-seq method. The DNA fragments were first converted by TET2 and APOBEC enzymes, followed by the single-strand DNA library construction. Briefly, tails and truncated adapter 1 were ligated to 3′ end of single-strand DNA. A new uracil-free DNA strand was generated through extension, and truncated adapter 2 was added to the other end of DNA. Finally, an indexing PCR step aimed to increase the yield of DNA molecules with full-length adapter 1 (P5) and adapter 2 (P7)