Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Methods Mol Biol. 2024;2842:405-418. doi: 10.1007/978-1-0716-4051-7_21.
DNA methylation is an important epigenetic modification that regulates chromatin structure and the cell-type-specific expression of genes. The association of aberrant DNA methylation with many diseases, as well as the increasing interest in modifying the methylation mark in a directed manner at genomic sites using epigenome editing for research and therapeutic purposes, increases the need for easy and efficient DNA methylation analysis methods. The standard approach to analyze DNA methylation with a single-cytosine resolution is bisulfite conversion of DNA followed by next-generation sequencing (NGS). In this chapter, we describe a robust, powerful, and cost-efficient protocol for the amplification of target regions from bisulfite-converted DNA, followed by a second PCR step to generate libraries for Illumina NGS. In the two consecutive PCR steps, first, barcodes are added to individual amplicons, and in the second PCR, indices and Illumina adapters are added to the samples. Finally, we describe a detailed bioinformatics approach to extract DNA methylation levels of the target regions from the sequencing data. Combining barcodes with indices enables a high level of multiplexing allowing to sequence multiple pooled samples in the same sequencing run. Therefore, this method is a robust, accurate, quantitative, and cheap approach for the readout of DNA methylation patterns at defined genomic regions.
DNA 甲基化是一种重要的表观遗传修饰，它调节染色质结构和基因的细胞类型特异性表达。异常的 DNA 甲基化与许多疾病有关，并且人们越来越有兴趣使用表观基因组编辑在基因组位点上以定向的方式修饰甲基化标记，用于研究和治疗目的，这增加了对简单有效的 DNA 甲基化分析方法的需求。以单个胞嘧啶分辨率分析 DNA 甲基化的标准方法是 DNA 的亚硫酸氢盐转化，随后是下一代测序（NGS）。在本章中，我们描述了一种强大、高效且具有成本效益的方法，用于扩增经亚硫酸氢盐转化的 DNA 中的靶区域，然后进行第二次 PCR 步骤以生成用于 Illumina NGS 的文库。在这两个连续的 PCR 步骤中，首先，将条形码添加到各个扩增子中，在第二个 PCR 中，将索引和 Illumina 接头添加到样品中。最后，我们描述了一种详细的生物信息学方法，用于从测序数据中提取靶区域的 DNA 甲基化水平。将条形码与索引相结合，可以实现高水平的多重化，从而能够在同一测序运行中对多个混合样本进行测序。因此，该方法是一种用于读取定义的基因组区域的 DNA 甲基化模式的可靠、准确、定量和廉价的方法。
