Enzymatic Methyl-seq (EM-seq): A Novel Approach for DNA Methylation Sequencing

Enzymatic Methyl-seq (EM-seq) [112] is the first methylation detection method to combine library preparation with oxidation, glycosylation, and deamination reactions, enabling simultaneous detection of 5mC and 5hmC. The method utilizes three enzymes: TET2, T4-BGT, and APOBEC3A. TET2 catalyzes the oxidation of 5mC to 5hmC, followed by further oxidation to 5fC and 5caC. T4-BGT catalyzes the glycosylation of 5hmC to 5-β-glucosyl-oxymethylcytosine (5gmC). APOBEC3A deaminates C and 5mC to U, while being insensitive to 5hmC, 5fC, 5caC, and 5gmC. TET2 is highly active, converting approximately 70% to 80% of 5mC to 5caC upon oxidation, with the remaining intermediate products, 5hmC and 5fC, accounting for approximately 10% each. This method ensures that 5mC and 5hmC are still recognized as C during Illumina sequencing, while unmodified C is converted to U and recognized as T during subsequent amplification and sequencing. Compared to bisulfite conversion, the enzymatic reaction significantly reduces the destruction of non-methylated C, improving 'GC coverage bias' due to the loss of GC-rich regions. Additionally, EM-seq requires a smaller starting amount of DNA, produces DNA libraries with longer and more complete insert fragments, and achieves higher mapping rates against the reference genome. However, this method simultaneously detects the methylation levels of both 5mC and 5hmC. To detect only 5mC, the results from the separate 5hmC detection should be excluded. Further research is needed to evaluate its market application.