Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EMseq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study—owing to their myogenic DMRs—overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage.

Bisulfite sequencing is widely used to detect 5mC and 5hmC at single base resolution. It is the most accepted method for detecting these cytosine modifications, but it does have significant drawbacks. DNA is frequently damaged resulting in fragmentation, loss of DNA and inherent biases introduced to sequencing data. To overcome this, we developed a new method called Enzymatic Methyl-seq (EMseq). This method relies on two sets of enzymatic reactions. In the first reaction, TET2 and T4-bGT convert 5mC and 5hmC into substrates that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines converting them to uracils. The protection of 5mC and 5hmC permits the discrimination of cytosines from 5mC and 5hmC. Over a range of DNA inputs, the overall fraction of 5mC and 5hmC in EM-seq libraries was similar to bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite converted libraries in all specificmeasures examined including coverage, duplication, sensitivity and nucleotide composition. EM-seq libraries displayed even GC distribution, improved correlation across input amounts, increased numbers of CpGs confidently assessed within genomic features, and improved the accuracy of cytosine methylation calls in other contexts. Bisulfite sequencing is known to severely damage DNA thus making library construction for lower DNA input very difficult. We show that EM-seq can be used to make libraries using as little as 100 pg of DNA. These libraries maintain all of the previously described advantages over bisulfite sequencing thus opening new avenues for research and clinical applications. Even with challenging input material, EM-seq provides a method to detect methylation state more reliably than WBGS.[7]. Sequencing distinguishes cytosines from these modified forms as they are read as thymines and cytosines respectively [8]. Despite its widespread use amongst epigenetic researchers, bisulfite sequencing also has significant drawbacks. It requires extreme temperatures and pH which causes depyrimidination of DNA resulting in DNA degradation [9]. Furthermore, cytosines are damaged disproportionately compared to 5mC or 5hmC. As a result, sequencing libraries made from converted DNA have an unbalanced nucleotide composition. All of these issues taken together result in libraries with reduced mapping rates and skewed GC bias plots, with a general under-representation of G-and Ccontaining dinucleotides and over-representation of AA-, AT-and TA-containing dinucleotides, when compared to a non-converted genome [10]. Therefore, the damaged libraries do not adequately cover the genome, and can include many gaps with little or no coverage. Increasing the sequencing depth of these libraries may recover some missing information, but at steep sequencing costs.These bisulfite library limitations have driven the development of new approaches for mapping 5mC and 5hmC, in combination or independently, for epigenome analysis. The methylation dependent restriction enzymes (MDRE), MspJI ...

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