DNA methylation analysis using long-read sequencing, methods and application

Abstract

Beyond the primary genetic code, DNA carries a second layer of information in the form of epigenetic modification, predominantly DNA methylation, which do not alter the DNA sequence itself but instead alter how genetic information is interpreted and expressed. DNA methylation is a dynamic process that can change in response to aging and environmental exposures, such as smoking. Importantly, altered DNA methylation patterns have been associated with a wide range of diseases, including cardiovascular disorders and cancer risk. Characterization of the role of DNA methylation is requisite on accurate genome-wide detection of methylation, however traditional approaches provide limited coverage of the genome and cannot accurately resolve parent-of-origin specific patterns. Long-read sequencing (LRS) overcomes these limitations and offers new opportunities to study DNA methylation. This thesis makes three main contributions. First, we establish LRS as highly reliable for DNA methylation detection and introduce filtering strategies to ensure high-quality data. Second, we reveal that sequencing variants drive much of the correlation of methylation with gene expression. Third, we map age associated methylation changes across the genome and reveal parent-of-origin specific changes of imprinting fidelity. Together, these studies demonstrate the utility of LRS for large scale methylation analysis and highlight its potential to uncover novel biological insights, refine our understanding of epigenetic regulation and inform future translational applications. Key words: DNA methylation, long-read sequencing, nanopore sequencing, gene regulation, methylation age