Profiling genome-wide DNA methylation

Abstract

DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

Keywords: Bisulfite sequencing; DNA methylation; Hydroxymethylation; Methylome; RRBS; Single-cell; WGBS.

Fig. 1

Commonly used methods for genome-wide DNA methylation analysis. a The procedures may involve fragmentation of genomic DNA by restriction enzyme digestion or sonication. The genomic DNA can be subjected to MBD enrichment, antibody enrichment, bisulfite conversion or TET oxidation before analyzing by microarray or next-generation sequencing platform. b Single-cell DNA methylation analysis that involves the isolation of single cells allows the assessment of methylation heterogeneity in cell populations while other genome-wide DNA methylation profiling methods using pooled heterogeneous cell populations are not capable to dissect the methylation heterogeneity. Blue concrete dots represent 5mC, and hollowed ones represent C. Each track represents 1 read

Fig. 2

Schematic overview of genome-wide DNA methylation profiling methods. a 5mC assays. b 5hmC assays. The actual sample requirement may vary according to the type of sample, genome size and number of PCR cycles

Fig. 3

Computational pipeline for genome-wide bisulfite sequencing data analysis. Reads from bisulfite sequencing are first aligned to the reference genome. The alignment data may be visualized in different tracks for comparison. After methylation calling, the bulk methylation level and genome-wide methylation level are calculated and plotted, and DMRs are determined. To perform an integrative analysis, DNA methylation data are coupled with gene expression, e.g., differentially expressed genes (DEGs), to delineate the regulatory role of DNA methylation