Epigenetic changes, such as DNA methylation, have been seen in various types of cancers, diseases, and neurological disorders. A common form of methylation occurs when a methyl group is added to the C-5 position of a cytosine, which is called 5-methyl-cyosine (5- mC). Hydroxymethylated is a type of methylation where 5mC is oxidized to 5-hydroxy-methyl- cytosine (5hmC), which has been linked to normal brain development. Current methods to sequence DNA methylation patterns (5-mC and 5-hmC) at a genome-wide level have several limitations, so there remains a need for a method to efficiently map DNA methylation. The state- of-the-art method for mapping DNA methylation uses bisulfite treatment, which shears DNA to small fragments, has a loss of genetic information due to unmethylated cytosines being converted to thymines, and has conversion error rates of about 1.5-1.6%. Moreover, this sequencing method takes substantial time and money as it requires a separate assay for genomic sequencing. Adaptations of this method have been made to distinguish 5-mC and 5-hmC, but they suffer from the same limitations. Nanopore sequencing can potentially overcome these challenges due to its ability to directly detect methylation and sequence long-reads. However, current nanopore sequencing techniques for methylation have high error rates. The scope of this thesis is multi- faceted, which involves implementing a methyl-cytosine and hydroxy-methyl-cytosine detection system by developing and benchmarking a new chemistry to modify cytosines to allow greater discrimination of unmodified and modified cytosines in a nanopore sequencer. The work of this thesis provides future researchers with a template to further increase the accuracy of detection between unmodified and modified cytosines in a nanopore sequencer by establishing an efficient, optimized protocol from the wet lab to the bioinformatic tools that were used
