一系列研究强调5-羟甲基脱氧胞嘧啶（5hmC）是一种重要的表观遗传标记。高分辨率的5hmC检测方法需要高测序深度，因此成本昂贵。许多研究使用富集法检测5hmC；然而，传统的富集法方法具有分辨率有限的缺点。为了克服这些局限性，我们开发了EBS-seq，一种在单碱基分辨率下结合高分辨率方法和富集法方法的低成本5hmC检测方法。EBS-seq使用选择性标记5hmC、脱氨基化胞嘧啶和5-甲基胞嘧啶、富集标记的5hmC以及DNA扩增期间进行C-to-T转化的方法。我们使用这种方法在HEK293T细胞和两个结直肠癌样本中分析了5hmC。与传统富集法检测5hmC相比，EBS-seq通过定位单个基对提高了5hmC信号的分辨率。此外，EBS-seq能够确定CpG岛和CpG海等CpG密集区域中的5hmC水平，避免了信号失真的问题。通过ACE-seq比较EBS-seq和传统高分辨率5hmC检测，我们发现EBS-seq更有效地发现5hmC位点。使用EBS-seq，我们发现基因表达和基因体内5hmC含量在HEK293T细胞和结直肠癌样本中均具有强烈的相关性。EBS-seq是一种可靠且成本低的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下。这种方法是在5hmC水平较低的具有挑战性的临床样本中进行5hmC检测的有前途的选择，比如癌症组织。©2023年作者。

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.© 2023. The Author(s).