Though a reference human genome and even excellent characterization of the epigenome (ENCODE, Epigenetics Roadmap) has been generated, the significance of noncoding genetic or epigenetic changes is still far from clear. With the advent of affordable DNA- sequencing technologies, methods have been developed for examining nuclear organization, chromatin state/histone post translation modifications, chromatin accessibility and methylation state. But these methods do not directly interrogate the DNA strand, and the reads are typically too short to provide critical correlative information. We propose the development of a novel epigenetic characterization methodology, fundamentally through the practical implementation of the sequencing of modified bases using a nanopore sequencing platform. Nanopore sequencing directly probes the chemical structure of the molecule in the pore with exquisite sensitivity. Its long reads enable correlation of epigenetic state over large (>10kb) stretches of the genome; each of these reads originates from a single cell, probing the epigenetic heterogeneity of the sample.
Epigenetics is the study of heritable changes in phenotype through a mechanism other than the direct genetic sequence. Sequencing of epigenetic marks is logistically difficult and limited with current 2nd generation technologies: only short-range interactions are studied, extensive chemical or enzymatic treatments are needed, and epigenetic heterogeneity is hard to probe. This proposal seeks to apply nanopore-based sequencing to characterization of the epigenetic state of the human genome via measurement of modifications to the DNA sequence.
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