Greally John M
Department of Genetics, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Ave., Bronx, NY, 10461, USA.
Methods Mol Biol. 2018;1708:191-207. doi: 10.1007/978-1-4939-7481-8_11.
Restriction enzymes have been valuable tools for representing the genome for DNA methylation assays, whether by using methylation-dependent enzymes or by sampling a reduced representation of the genome using a methylation-insensitive enzyme. These survey assays have remained mainstays of genome-wide approaches even with the development of more comprehensive shotgun genome-wide bisulphite sequencing-based assays, as they are significantly more affordable. DNA methylation survey assays are numerous and include reduced representation bisulphite sequencing (RRBS), the Illumina HumanMethylation450K and EPIC microarray system, and our evolving series of HELP-based assays. The HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assays initially involved microarray-based reporting of DNA methylation, but have now migrated to the use of massively parallel sequencing. In this chapter, we describe the latest HELP-tagging assay that uses Illumina Tru-Seq adapters, and mention the extension of the HELP-tagging assay to quantify 5-hydroxymethylation using the HELP-GT assay.
无论是使用依赖甲基化的酶，还是使用对甲基化不敏感的酶对基因组进行抽样以获得简化的基因组表示，限制性内切酶一直都是用于DNA甲基化检测的基因组表示的重要工具。即使随着基于全基因组亚硫酸氢盐测序的更全面的鸟枪法检测方法的发展，这些检测方法仍然是全基因组方法的主要手段，因为它们的成本要低得多。DNA甲基化检测方法众多，包括简化代表性亚硫酸氢盐测序（RRBS）、Illumina HumanMethylation450K和EPIC微阵列系统，以及我们不断发展的基于HELP的检测方法系列。HELP（通过连接介导的PCR富集HpaII小片段）检测最初涉及基于微阵列的DNA甲基化报告，但现在已转向使用大规模平行测序。在本章中，我们描述了使用Illumina Tru-Seq接头的最新HELP标记检测方法，并提及了HELP标记检测方法的扩展，即使用HELP-GT检测方法来量化5-羟甲基化。
