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. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

中文翻译：

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酶促甲基测序以单碱基分辨率从皮克 DNA 中检测 DNA 甲基化

亚硫酸氢盐测序以单碱基分辨率检测 5mC 和 5hmC。然而，亚硫酸氢盐处理会损害 DNA，从而导致片段化、DNA 丢失和测序数据有偏差。为了克服这些问题，开发了酶甲基测序（EM-seq）。该方法使用两组酶促反应检测 5mC 和 5hmC。在第一个反应中，TET2 和 T4-BGT 将 5mC 和 5hmC 转化为不能被 APOBEC3A 脱氨基的产物。在第二个反应中，APOBEC3A 通过将未修饰的胞嘧啶转化为尿嘧啶来脱氨。因此，这三种酶能够识别5mC和5hmC。将 EM-seq 文库与亚硫酸氢盐转化的 DNA 进行比较，并且在转化之前将每种文库类型连接至 Illumina 接头。使用 NA12878 基因组 DNA、游离 DNA 和 FFPE DNA 在一系列 DNA 输入上构建文库。EM-seq 文库中检测到的 5mC 和 5hmC 与亚硫酸氢盐文库中检测到的相似。然而，在所有检查的具体指标（覆盖率、重复、敏感性等）中，使用 EM-seq 构建的文库均优于亚硫酸氢盐转化的文库。EM-seq 文库显示出均匀的 GC 分布、DNA 输入之间更好的相关性、基因组特征中 CpG 数量的增加以及胞嘧啶甲基化调用的准确性。EM-seq 只需 100 pg DNA 即可有效，并且这些文库相对于亚硫酸氢盐测序保持了所描述的优势。EM-seq 文库构建使用具有挑战性的样本和较低的 DNA 输入量，为研究和临床应用开辟了新途径。