Zhang Baolong, Li Wei, Li Jin, Li Yan, Luo Huaibing, Xi Yanping, Dong Shihua, Wu Feizhen, Yu Wenqiang
Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.
Key Laboratory of Medical Epigenetics and Metabolism, Institute of Clinical Science of Zhongshan Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Front Cell Dev Biol. 2024 Sep 24;12:1457387. doi: 10.3389/fcell.2024.1457387. eCollection 2024.
Whole-genome bisulfite sequencing (WGBS) has been extensively utilized for DNA methylation profiling over the past decade. However, it has shown limitations in terms of high costs and inefficiencies. The productivity and accuracy of DNA methylation detection rely critically on the optimization of methodologies and the continuous refinements of related sequencing platforms. Here, we describe a detailed protocol of guide positioning sequencing (GPS), a bisulfite-based, location-specific sequencing technology designed for comprehensive DNA methylation characterization across the genome. The fundamental principle of GPS lies in the substitution of dCTP with 5-methyl-dCTP (5 mC) at the 3'-end of DNA fragments by T4 DNA polymerase, which protects cytosines from bisulfite conversion to preserve the integrity of the base composition. This alteration allows the 3'-end to independently facilitate genetic variation profiling and guides the 5'-end, enriched with methylation information, to align more rapidly to the reference genome. Hence, GPS enables the concurrent detection of both genetic and epigenetic variations. Additionally, we provide an accessible description of the data processing, specifically involving certain software and scripts. Overall, the entire GPS procedure can be completed within a maximum of 15 days, starting with a low initial DNA input of 100-500 ng, followed by 4-5 days for library construction, 8-10 days for high-throughput sequencing (HTS) and data analysis, which can greatly facilitate the promotion and application of DNA methylation detection, especially for the rapid clinical diagnosis of diverse disease pathologies associated with concurrent genetic and epigenetic variations.
在过去十年中，全基因组亚硫酸氢盐测序（WGBS）已被广泛用于DNA甲基化分析。然而，它在成本高和效率低方面存在局限性。DNA甲基化检测的效率和准确性在很大程度上依赖于方法的优化和相关测序平台的不断改进。在此，我们描述了一种详细的引导定位测序（GPS）方案，这是一种基于亚硫酸氢盐的、针对全基因组全面DNA甲基化特征分析设计的位点特异性测序技术。GPS的基本原理在于通过T4 DNA聚合酶在DNA片段的3'端用5-甲基-dCTP（5mC）替代dCTP，这可保护胞嘧啶不被亚硫酸氢盐转化，从而保持碱基组成的完整性。这种改变使3'端能够独立地促进遗传变异分析，并引导富含甲基化信息的5'端更快地比对到参考基因组。因此，GPS能够同时检测遗传和表观遗传变异。此外，我们还提供了数据处理的详细说明，特别是涉及某些软件和脚本。总体而言，整个GPS流程最多可在15天内完成，起始DNA输入量低至100 - 500 ng，随后文库构建需要4 - 5天，高通量测序（HTS）和数据分析需要8 - 10天，这可极大地促进DNA甲基化检测的推广和应用，特别是用于与同时存在的遗传和表观遗传变异相关的多种疾病病理的快速临床诊断。
