Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide–primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.

5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）是氧化DNA去甲基化通路中最普遍的中间产物，参与调控胚胎发生、神经过程与癌变过程。对临床样本中该相对稀缺的基因组修饰进行谱分析，需要采用无需严苛化学处理的低成本高分辨率技术。本研究报道一种可实现单核苷酸分辨率下5hmC谱分析的无亚硫酸氢盐方法——hmTOP-seq（5hmC特异性锚定寡核苷酸引发测序，5hmC-specific tethered oligonucleotide–primed sequencing），其原理是从原位锚定的DNA寡核苷酸处，对共价标记的5hmC位点启动直接测序读长。通过对不同偶联化学体系的研究，我们提出了锚定导向的非同源聚合酶引发结构模型，可在方法设计阶段对适配的锚定物开展理论评估。我们在小型模式基因组与小鼠胚胎干细胞中对hmTOP-seq流程进行了优化与验证，借此构建了单核苷酸分辨率的5hmC图谱，可反映链特异性CG羟甲基化的细微差异。综上，hmTOP-seq为低成本、精准识别5hmC提供了全新的宝贵工具，可为解析其生物学功能以及与人类疾病相关的表观遗传变化提供重要支撑。