Comparison of current methods for genome-wide DNA methylation profiling

DNA methylation is an epigenetic mechanism involved in gene regulation and cellular differentiation. Accurate and comprehensive assessment of DNA methylation patterns is thus essential for understanding their role in various biological processes and disease mechanisms. Bisulfite sequencing has long been the default method for analyzing methylation marks due to its single-base resolution, but the associated DNA degradation poses a concern. Although several methods have been proposed to circumvent this issue, there is no clear consensus on which method might be better suited for specific study designs. We conducted a comparative evaluation of four DNA methylation detection approaches: whole-genome bisulfite sequencing (WGBS), Illumina methylation microarray (EPIC), enzymatic methyl-sequencing (EM-seq) and third-generation sequencing by Oxford Nanopore Technologies (ONT). DNA methylation profiles were assessed across three human genome samples derived from tissue, cell line, and whole blood. We systematically compared these methods in terms of resolution, genomic coverage, methylation calling accuracy, cost, time, and practical implementation. EM-seq showed the highest concordance with WGBS, indicating strong reliability due to their similar sequencing chemistry. ONT sequencing, while showing lower agreement with WGBS and EM-seq, captured certain loci uniquely and enabled methylation detection in challenging genomic regions. Despite a substantial overlap in CpG detection among methods, each method identified unique CpG sites, emphasizing their complementary nature. Our findings underscore the strengths and limitations of current DNA methylation detection methods. EM-seq and ONT emerge as robust alternatives to WGBS and EPIC, offering unique advantages: EM-seq delivers consistent and uniform coverage, while ONT excels in long-range methylation profiling and access to challenging genomic regions. These insights provide practical guidance for method selection based on specific experimental goals.