ABSTRACT

Genome-wide DNA methylation analysis is one of the most common epigenetic processes analysed for genome characterization and differential DNA methylation assessment. Previous genome-wide analysis has suggested an important variable in DNA methylation methods involves CpG density. The current study was designed to investigate the CpG density in a variety of different species genomes and correlate this to various DNA methylation analysis data sets. The majority of all genomes had >90% of the genome in the low density 1–3 CpG/100 bp category, while <10% of the genome was in the higher density >5 CpG/100 bp category. Similar observations with human, rat, bird, and fish genomes were observed. The methylated DNA immunoprecipitation (MeDIP) procedure uses the anti-5-methylcytosine antibody immunoprecipitation followed by next-generation sequencing (MeDIP-Seq). The MeDIP procedure is biased to lower CpG density of <5 CpG/100 bp, which corresponds to >95% of the genome. The reduced representation bisulphite (RRBS) protocol generally identifies DMRs in higher CpG density regions of ≥3 CpG/100 bp which corresponds to approximately 20% of the genome. The whole-genome bisulphite (WGBS) analyses resulted in higher CpG densities, often greater than 10 CpG/100bp. WGBS generally identifies ≥2 CpG/100bp, which corresponds to approximately 50% of the genome. Limitations and potential optimization approaches for each method are discussed. None of the procedures can provide complete genome-wide assessment of the genome, but MeDIP-Seq provides coverage of the highest percentage. Observations demonstrate that CpG density is a critical variable in DNA methylation analysis, and different molecular techniques focus on distinct genomic regions.

摘要

全基因组 DNA 甲基化分析是用于基因组表征和差异 DNA 甲基化评估的最常见的表观遗传过程之一。以前的全基因组分析表明 DNA 甲基化方法中的一个重要变量涉及 CpG 密度。目前的研究旨在调查各种不同物种基因组中的 CpG 密度，并将其与各种 DNA 甲基化分析数据集相关联。所有基因组中的大多数都具有 >90% 的基因组处于低密度 1-3 CpG/100 bp 类别中，而 <10% 的基因组处于较高密度 >5 CpG/100 bp 类别中。观察到人类、大鼠、鸟类和鱼类基因组的类似观察结果。甲基化 DNA 免疫沉淀 (MeDIP) 程序使用抗 5-甲基胞嘧啶抗体免疫沉淀，然后进行下一代测序 (MeDIP-Seq)。MeDIP 程序偏向于 <5 CpG/100 bp 的较低 CpG 密度，这对应于 >95% 的基因组。减少代表性亚硫酸氢盐 (RRBS) 协议通常在≥3 CpG/100 bp 的较高 CpG 密度区域中识别 DMR，这对应于大约 20% 的基因组。全基因组亚硫酸氢盐 (WGBS) 分析导致更高的 CpG 密度，通常大于 10 CpG/100bp。WGBS 通常识别≥2 CpG/100bp，对应于大约 50% 的基因组。讨论了每种方法的局限性和潜在的优化方法。没有一个程序可以提供对基因组的完整全基因组评估，但 MeDIP-Seq 提供了最高百分比的覆盖率。观察表明，CpG 密度是 DNA 甲基化分析中的一个关键变量，不同的分子技术侧重于不同的基因组区域。