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Methylation microarray and bisulphite sequencing are often used to study 5’-methylcytosine (5’-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5’-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5’-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (&gt;0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5’-mC in the human genome with low coverage depth.

Whole human genome 5’-mC methylation analysis using long read nanopore sequencing