Combining MeDIP-seq and MRE-seq to investigate genome-wide CpG methylation.
Combining MeDIP-seq and MRE-seq to investigate genome-wide CpG methylation.
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DOI：
10.1016/j.ymeth.2014.10.032
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发表时间：
2015-01-15
期刊：
影响因子：
4.8
通讯作者：
Wang, Ting
中科院分区：
文献类型：
--
作者：
Li, Daofeng;Zhang, Bo;Xing, Xiaoyun;Wang, Ting
DNA CpG甲基化是包括哺乳动物在内的高等真核生物中广泛存在的表观遗传标记。DNA甲基化在多种生物学过程中起着关键作用，如X染色体失活、转座因子抑制、基因组印记和基因表达控制。基于测序的DNA甲基化分析方法的最新进展为以全基因组方式测量DNA甲基化提供了前所未有的机会，使得全面研究DNA甲基化的作用成为可能。已经开发了几种方法，例如全基因组亚硫酸氢盐测序（WGBS）、还原型亚硫酸氢盐测序（RRBS）和基于富集的方法，包括甲基化依赖性免疫沉淀随后测序甲基-CpG结合结构域（MBD）蛋白富集的基因组测序（MBD-seq），甲基转移酶指导的活化基团转移随后测序（mTAG），和甲基化敏感性限制性内切酶消化后测序（MRE-seq）。这些方法的不同之处在于它们的基因组CpG覆盖率、分辨率、定量准确性、成本和用于分析数据的软件。其中，WGBS被认为是黄金标准。然而，由于所需的测序深度，对于典型的实验室来说，它仍然是一种成本高昂的技术。我们发现，通过整合两种本质上互补的基于富集的方法（即，MeDIP-seq和MRE-seq），我们可以显著提高全DNA甲基化组谱分析的效率。通过使用两个最近开发的计算算法（即，M&M和methylCRF），MeDIP-seq和MRE-seq的组合产生高覆盖率和高分辨率的全基因组CpG甲基化测量，以及差异甲基化区域的稳健预测。因此，两种基于富集的方法的组合提供了WGBS的具有成本效益的替代方案。在这篇文章中，我们描述了用于执行MeDIP-seq和MRE的实验协议，以及用于运行M&M和methylCRF的计算协议。
DNA CpG methylation is a widespread epigenetic mark in high eukaryotes including mammals. DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and control of gene expression. Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to measure DNA methylation in a genome-wide fashion, making it possible to comprehensively investigate the role of DNA methylation. Several methods have been developed, such as Whole Genome Bisulfite Sequencing (WGBS), Reduced Representation Bisulfite Sequencing (RRBS), and enrichment-based methods including Methylation Dependent ImmunoPrecipitation followed by sequencing (MeDIP-seq), methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), methyltransferase-directed Transfer of Activated Groups followed by sequencing (mTAG), and Methylation-sensitive Restriction Enzyme digestion followed by sequencing (MRE-seq). These methods differ by their genomic CpG coverage, resolution, quantitative accuracy, cost, and software for analyzing the data. Among these, WGBS is considered the gold standard. However, it is still a cost-prohibitive technology for a typical laboratory due to the required sequencing depth. We found that by integrating two enrichment-based methods that are complementary in nature (i.e., MeDIP-seq and MRE-seq), we can significantly increase the efficiency of whole DNA methylome profiling. By using two recently developed computational algorithms (i.e., M&M and methylCRF), the combination of MeDIP-seq and MRE-seq produces genome-wide CpG methylation measurement at high coverage and high resolution, and robust predictions of differentially methylated regions. Thus, the combination of the two enrichment-based methods provides a cost-effective alternative to WGBS. In this article we describe both the experimental protocols for performing MeDIP-seq and MRE, and the computational protocols for running M&M and methylCRF.
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