Department of Chemistry, Yonsei University, Seoul, Korea.
IMBdx, Seoul, Korea.
Clin Epigenetics. 2023 Mar 1;15(1):34. doi: 10.1186/s13148-023-01451-7.
A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods.
EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples.
EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues.
越来越多的研究强调 5-羟甲基胞嘧啶（5hmC）作为一种重要的表观遗传标记。高分辨率检测 5hmC 的方法需要高测序深度，因此成本高昂。许多研究使用基于富集的方法来检测 5hmC；然而，传统的基于富集的方法分辨率有限。为了克服这些限制，我们开发了 EBS-seq，这是一种具有单碱基分辨率的高效检测 5hmC 的方法，它结合了高分辨率方法和基于富集的方法的优势。
EBS-seq 使用 5hmC 的选择性标记、胞嘧啶和 5-甲基胞嘧啶的脱氨、标记的 5hmC 的下拉以及 DNA 扩增过程中的 C 到 T 转换。使用这种方法，我们对 HEK293T 细胞和两个结直肠癌样本进行了 5hmC 谱分析。与传统的基于富集的 5hmC 检测相比，EBS-seq 通过将信号定位在单碱基分辨率上来提高 5hmC 信号。此外，EBS-seq 能够确定在信号可能发生扭曲的 CpG 密集区域（如 CpG 岛和 CpG 侧翼）中的 5hmC 水平。通过 ACE-seq 将 EBS-seq 与传统的高分辨率 5hmC 检测进行比较，我们表明 EBS-seq 更有效地发现 5hmC 位点。使用 EBS-seq，我们在 HEK293T 细胞和结直肠癌样本中发现了基因表达与基因体 5hmC 含量之间的强关联。
EBS-seq 是一种可靠且经济高效的 5hmC 检测方法，因为它同时富集含有 5hmC 的 DNA 片段，并将 5hmC 信号定位在单碱基分辨率。这种方法是在具有低 5hmC 水平的挑战性临床样本（如癌症组织）中进行 5hmC 检测的有前途的选择。
