Comparison of nanopore sequencing, MethylationEPIC array, and EM-Seq for DNA methylation detection

DNA methylation is an important biological process in epigenetics, and many methods have been developed to profile DNA methylation. Recent studies have employed Oxford Nanopore long-read sequencing for DNA methylation detection, presenting an alternative to the widely utilized Infinium arrays and short-read methods. In this study, we evaluate the performance of Nanopore sequencing in DNA methylation detection by comparing it to Illumina's MethylationEPIC microarray (EPIC) and Enzymatic Methyl-Sequencing (EM-Seq). The initial comparison was between Nanopore and the EPIC array using blood samples (n = 4). Among the ∼850,000 CpG sites covered by both methods, we observed high concordance (Pearson correlation coefficient, r ≥ 0.94 across all four samples). After downsampling Nanopore data from an average coverage of 26.4 reads per site to 10 reads per site, the correlation in CpG methylation remained high (r ≥ 0.93). When comparing Nanopore and EM-Seq using brain tissue samples (n = 4), lower correlation of CpG methylation (r: 0.79-0.88) was detected between Nanopore and EM-Seq, which can be attributed to biased and reduced coverage of hypomethylated CpG sites by EM-Seq. We also investigated additional features of Nanopore sequencing, such as native DNA sequencing that can differentiate 5mC and 5hmC, as well as haplotype phasing. Overall, the Nanopore platform exhibited a high degree of concordance with the EPIC array and provided more uniform genomic coverage than EM-Seq. This study provides insights for researchers in selecting appropriate DNA methylation detection methods, considering factors such as cost, DNA input, and the complexity of downstream analysis.