5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human–mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.

5-甲基胞嘧啶（5mC）是表观遗传修饰的重要类型。亚硫酸氢盐测序（BS-seq）有局限性，例如DNA严重降解。使用单分子实时测序，我们开发了一种直接检查5mC的方法。该方法从整体上检查了DNA聚合酶的动力学信号（包括脉冲间持续时间和脉冲宽度）以及测量窗口内每个核苷酸的序列背景（称为整体动力学（HK）模型）。每个分析的双链DNA分子的测量窗口均包含21个核苷酸，中心的CpG位点带有一个胞嘧啶。我们使用了扩增的DNA（未甲基化）和经M.SssI处理的DNA（甲基化）（M.SssI是CpG甲基转移酶）来训练卷积神经网络。使用此类样品区分甲基化状态的曲线下面积最大为0.97。在单碱基分辨率下，全基因组5mC检测的灵敏度和特异性分别达到90％和94％。然后，在人-鼠杂种片段上测试了HK模型，其中杂种的每个成员具有不同的甲基化状态。还对从各种生物样品（如血沉棕黄层，胎盘和肿瘤组织）提取的人类基因组DNA分子进行了测试。HK模型推导的总体甲基化水平与BS-seq（然后，在人-鼠杂种片段上测试了HK模型，其中杂种的每个成员具有不同的甲基化状态。还对从各种生物样品（如血沉棕黄层，胎盘和肿瘤组织）提取的人类基因组DNA分子进行了测试。HK模型推导的总体甲基化水平与BS-seq（然后，在人-鼠杂种片段上测试了HK模型，其中杂种的每个成员具有不同的甲基化状态。还对从各种生物样品（如血沉棕黄层，胎盘和肿瘤组织）提取的人类基因组DNA分子进行了测试。HK模型推导的总体甲基化水平与BS-seq（r = 0.99；P <0.0001），并允许测量印迹基因中的等位基因特异性甲基化模式。综上所述，该方法提供了同时进行全基因组遗传和表观遗传学分析的系统。