Nat. Biotechnol. https://doi.org/10.1038/nbt.4204 (2018)

5-Methylcytosine (5mC) undergoes DNA demethylation through TET-mediated oxidation, in the process forming the stable intermediate 5-hydroxymethylcytosine (5hmC), which is enriched in neurons and embryonic stem cells. Detection of 5hmC at single-base resolution relies on adapting sequencing methods that utilize bisulfite to chemically convert cytosine and 5mC to thymidine but leave 5hmC protected from conversion. However, bisulfite also induces significant degradation of genomic DNA, preventing analysis of rare cell types. To address this limitation, Schutsky et al. developed an enzymatic method called APOBEC-coupled epigenetic sequencing (ACE-seq). Building on prior biochemical evidence that AID/APOBEC DNA deaminases can discriminate between modified cytosine bases, they utilized APOBEC3A to enzymatically convert cytosine and 5mC, but not 5hmC bases. Testing of ACE-seq on phage genomes showed accurate detection of >98.5% of 5hmC bases, with cytosine and 5mC nonconversion rates of <0.5%, outperforming existing bisulfite-dependent methods without impacting DNA stability. ACE-seq analysis of purified cortical excitatory neurons revealed that specific genomic features can differ greatly in 5hmC content, with imprinted regions depleted for 5hmC and enhancers highly enriched. Overall, ACE-seq provides a reliable new method to detect 5-hmC with high confidence.