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.

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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Nucleosome occupancy plays a key role in regulating access to eukaryotic genomes. Although various chromatin regulatory complexes are known to regulate nucleosome occupancy, the role of DNA sequence in this regulation remains unclear, particularly in mammals. To address this problem, we measured nucleosome distribution at high temporal resolution in human cells at hundreds of genes during the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV). We show that nucleosome redistribution peaks at 24 h post-KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro-assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 h post-KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and ''spring'' to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans.

The development and progression of lung adenocarcinoma, one of the most common cancers, is driven by the interplay of genetic and epigenetic changes and the role of chromatin structure in malignant transformation remains poorly understood. We used systematic nucleosome distribution and chromatin accessibility microarray mapping platforms to analyze the genome-wide chromatin structure from normal tissues and from primary lung adenocarcinoma of different grades and stages. We identified chromatin-based patterns across different patients with lung adenocarcinoma of different cancer grade and stage. Low-grade cancers had nucleosome distributions very different compared with the corresponding normal tissue but had nearly identical chromatin accessibility. Conversely, nucleosome distributions of high-grade cancers showed few differences. Substantial disruptions in chromosomal accessibility were seen in a patient with a high-grade and high-stage tumor. These data imply that chromatin structure changes during the progression of lung adenocarcinoma. We have therefore developed a model in which low-grade lung adenocarcinomas are linked to changes in nucleosome distributions, whereas higher-grade tumors are linked to large-scale chromosomal changes. These results provide a foundation for the development of a comprehensive framework linking the general and locus-specific roles of chromatin structure to lung cancer progression. We propose that this strategy has the potential to identify a new class of chromatin-based diagnostic, prognostic and therapeutic markers in cancer progression.