Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are modified versions of cytosine in DNA with roles in regulating gene expression. Using whole genomic DNA from mouse cerebellum, we have benchmarked 5mC and 5hmC detection by nanopore sequencing against oxidative bisulphite sequencing (oxBS-seq) and TET-assisted bisulphite sequencing (TAB-seq). We further examine the use of nanopore duplex modified base detection to study strand-specific asymmetrical modifications. Nanopore detection of 5mC and 5hmC is highly consistent with orthologous oxBS-seq (RMSD 19.45%) and TAB-seq (RMSD 15.48%) and outperforms these methods in reproducibility. We note a high level of asymmetric methylation (21.1 % of base-calls) for a non-dividing tissue; however, context-specific rates of symmetrical modification were found at differentially methylated regions in imprinted genes, as well as at CTCF binding sites, suggesting biological importance for maintaining modification fidelity within these regions. This study demonstrates the unique ability of nanopore sequencing to improve the efficiency, resolution, and detail of cytosine modification mapping.