Department of Ecology, Evolution and Environmental Biology, Columbia University, New York, New York, United States of America.
Center for Integrative Animal Behavior, Columbia University, New York, New York, United States of America.
PLoS One. 2023 Mar 9;18(3):e0282672. doi: 10.1371/journal.pone.0282672. eCollection 2023.
The increasing interest in studying DNA methylation to understand how traits or diseases develop requires new and flexible approaches for quantifying DNA methylation in a diversity of organisms. In particular, we need efficient yet cost-effective ways to measure CpG methylation states over large and complete regions of the genome. Here, we develop TEEM-Seq (target-enriched enzymatic methyl sequencing), a method that combines enzymatic methyl sequencing with a custom-designed hybridization capture bait set that can be scaled to reactions including large numbers of samples in any species for which a reference genome is available. Using DNA from a passerine bird, the superb starling (Lamprotornis superbus), we show that TEEM-Seq is able to quantify DNA methylation states similarly well to the more traditional approaches of whole-genome and reduced-representation sequencing. Moreover, we demonstrate its reliability and repeatability, as duplicate libraries from the same samples were highly correlated. Importantly, the downstream bioinformatic analysis for TEEM-Seq is the same as for any sequence-based approach to studying DNA methylation, making it simple to incorporate into a variety of workflows. We believe that TEEM-Seq could replace traditional approaches for studying DNA methylation in candidate genes and pathways, and be effectively paired with other whole-genome or reduced-representation sequencing approaches to increase project sample sizes. In addition, TEEM-Seq can be combined with mRNA sequencing to examine how DNA methylation in promoters or other regulatory regions is related to the expression of individual genes or gene networks. By maximizing the number of samples in the hybridization reaction, TEEM-Seq is an inexpensive and flexible sequence-based approach for quantifying DNA methylation in species where other capture-based methods are unavailable or too expensive, particularly for non-model organisms.
越来越多的人对研究 DNA 甲基化以了解性状或疾病的发展机制产生了兴趣，这需要新的、灵活的方法来量化各种生物中 DNA 甲基化的程度。特别是，我们需要高效且具有成本效益的方法来测量基因组大片段和完整区域中的 CpG 甲基化状态。在这里，我们开发了 TEEM-Seq（靶向富集酶甲基化测序），这是一种将酶甲基化测序与定制设计的杂交捕获探针集相结合的方法，可扩展到包括任何具有参考基因组的物种的大量样本的反应中。我们使用一种雀形目鸟类，即华丽八哥（Lamprotornis superbus）的 DNA 进行实验，结果表明 TEEM-Seq 能够与全基因组和简化基因组测序等更为传统的方法一样准确地定量 DNA 甲基化状态。此外，我们还证明了其可靠性和可重复性，因为来自相同样本的重复文库高度相关。重要的是，TEEM-Seq 的下游生物信息学分析与任何基于序列的 DNA 甲基化研究方法相同，使其易于整合到各种工作流程中。我们相信，TEEM-Seq 可以替代候选基因和途径中研究 DNA 甲基化的传统方法，并与其他全基因组或简化基因组测序方法有效地结合，以增加项目样本量。此外，TEEM-Seq 可以与 mRNA 测序相结合，研究启动子或其他调控区域的 DNA 甲基化如何与单个基因或基因网络的表达相关。通过最大限度地增加杂交反应中的样本数量，TEEM-Seq 是一种经济实惠且灵活的基于序列的方法，可用于量化其他捕获方法不可用或过于昂贵的物种中的 DNA 甲基化程度，特别是对于非模式生物。
