Date of Award

Summer 8-17-2023

Author's School

McKelvey School of Engineering

Author's Department

Biomedical Engineering

Degree Name

Master of Science (MS)

Degree Type



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.


English (en)


John R. Edwards, Washington University School of Medicine

Committee Members

Guy Genin, Jianmin Cui

Available for download on Wednesday, July 10, 2024