NEBNEXT® Enzymatic Methyl-seq Workflow

A workflow animation for an enzyme-based method for detection of 5mC and 5hmC, an alternative to bisulfite sequencing.


The EM-seq workflow is a new enzyme-based method for detection of 5-methylcytosine and 5-hydroxymethylcytosine at the single base level within DNA. This method minimizes damage to DNA and provides superior performance compared to bisulfite sequencing, including more sensitive detection of 5mC and 5hmC, uniform GC coverage, greater mapping efficiency, and detection of more CpGs with fewer sequence reads. The workflow begins with DNA that has been mechanically sheared. This DNA is end repaired, dA-tailed and ligated to the EM-seq Adaptor using NEBNext Ultra II reagents. The DNA is now ready to move into the EM-seq conversion reactions. The EM-seq conversion reaction is a two-step process: The first enzymatic step involves the oxidation of 5-methylcytosine to 5-carboxycytosine using TET2 and the glucosylation of 5-hydroxymethylcytosine using the Oxidation Enhancer. These reactions protect these bases from deamination in the next step. Before the second enzymatic step, the DNA is made single-stranded using either formamide or sodium hydroxide. APOBEC then deaminates cytosines into uracils. Because 5-methylcytosines and 5-hydroxymethylcytosines were protected in the first enzymatic step, these forms are no longer substrates for APOBEC and are not deaminated. The last stage of EM-seq library construction is PCR. The EM-seq libraries are amplified using NEBNext Q5U. This is a modified form of NEBNext Q5 polymerase that can amplify uracil- containing templates, and in EM-seq the primers used for amplification contain unique dual indexes, and the amplified libraries are compatible with Illumina sequencing. For bioinformatic analysis, pipelines used for bisulfite sequencing can be used for EM-seq.
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