Indirect approaches to measure methylation include pull-down assays with methylation-specific antibodies and methyl-binding proteins, and restriction digestion with enzymes with preferences for or against methylcytosines. The first step in the study of DNA methylation is to determine whether or not a given cytosine residue is methylated. DNA methylation is involved in a large number of cellular processes including genomic imprinting, X chromosome inactivation, embryonic development, and transcriptional regulation of developmentally important genes, as well as in ensuring genome integrity and protecting against invasive DNAs. DNA methylation is typically associated with the silencing of genes and repetitive DNAs such as transposable elements however, expressed genes are also found to be methylated. CHH methylation is also called asymmetrical methylation. Non-CG methylation, where modification occurs in CHG and CHH contexts (where H corresponds to A, T, or C residues), occurs in plants and many other organisms. DNA methylation that takes place at cytosine residues which are followed by guanine is termed CG methylation and is conserved in most eukaryotes. The fifth carbon position of cytosine in DNA can be covalently modified by the addition of a methyl group to form 5-methylcytosine (5-mC). It may therefore be more desirable to use EM-seq in methylation studies. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. We suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines.
Using an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. 5′ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes.
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