DNA methylation is a well-known epigenetic modification that plays a crucial role in gene regulation, but genome-wide analysis of DNA methylation remains technically challenging and costly. DNA methylation-dependent restriction enzymes can be used to restrict CpG methylation analysis to methylated regions of the genome only, which significantly reduces the required sequencing depth and simplifies subsequent bioinformatics analysis. Unfortunately, this approach has been hampered by complete digestion of DNA in CpG methylation-dense regions, resulting in fragments that are too small for accurate mapping. Here, we show that the activity of DNA methylation-dependent enzyme, LpnPI, is blocked by a fragment size smaller than 32 bp. This unique property prevents complete digestion of methylation-dense DNA and allows accurate genome-wide analysis of CpG methylation at single-nucleotide resolution. Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments revealed highly reproducible genome-wide CpG methylation profiles for >50% of all potentially methylated CpGs, at a sequencing depth less than one-tenth required for whole-genome bisulfite sequencing (WGBS). MeD-seq identified a high number of patient and tissue-specific differential methylated regions (DMRs) and revealed that patient-specific DMRs observed in both blood and buccal samples predict DNA methylation in other tissues and organs. We also observed highly variable DNA methylation at gene promoters on the inactive X Chromosome, indicating tissue-specific and interpatient-specific escape of X Chromosome inactivation. These findings highlight the potential of MeD-seq for high-throughput epigenetic profiling.

DNA甲基化是众所周知的表观遗传修饰，在基因调控中起着至关重要的作用，但是DNA甲基化的全基因组分析在技术上仍然具有挑战性且成本高昂。DNA甲基化依赖性限制性内切酶可用于仅将CpG甲基化分析限制在基因组的甲基化区域，从而大大减少了所需的测序深度并简化了后续的生物信息学分析。不幸的是，这种方法已被CpG甲基化密集区中DNA的完全消化所阻碍，导致片段太小而无法精确定位。在这里，我们显示了DNA甲基化依赖性酶LpnPI的活性被小于32 bp的片段所阻断。这种独特的特性可防止甲基化致密DNA的完全消化，并允许以单核苷酸分辨率对CpG甲基化进行全基因组范围的准确分析。LpnPI酶切片段的甲基化DNA测序（MeD-seq）显示了> 50％的潜在甲基化CpG的高度可重现的全基因组CpG甲基化谱图，其测序深度不到全基因组亚硫酸氢盐测序（WGBS）所需十分之一。 。MeD-seq识别出大量的患者和组织特异性甲基化差异区域（DMR），并揭示了在血液和颊样品中观察到的患者特异性DMR可以预测其他组织和器官中的DNA甲基化。我们还观察到非活性X染色体上基因启动子处的DNA甲基化高度可变，表示X染色体失活的组织特异性和患者特异性逃逸。这些发现凸显了MeD-seq在高通量表观遗传学分析中的潜力。