Direct detection of 8-oxo-dG using nanopore sequencing

Abstract

Genomic DNA is constantly subjected to oxidative damage, which is thought to be one of the major drivers of cancer and age-dependent decline. The most prominent consequence is the modification of guanine into 8-hydroxyguanine (8-oxo-dG), which has important mutagenic potential and plays a role in methylation-mediated gene regulation. Methods to simultaneously detect and quantify 8-oxo-dG within its genomic context have been lacking; mainly because these methods rely on indirect detection or are based on hydrolysis of the DNA. Nanopore sequencing has been deployed for the direct detection of base-modifications like cytosine methylation during sequencing. However, currently there is no model to detect 8-oxo-dG by nanopore sequencing due to the lack of training data. Here, we developed a strategy based on synthetic oligos to create long DNA molecules with context variability for effective deep learning and nanopore sequencing. Moreover, we showcase a training approach suitable to deal with the extreme scarceness of 8-oxo-dG compared to canonical G to enable specific 8-oxo-dG detection. Applied to an inducible tissue culture system for oxidative DNA damage, our approach reveals variable 8-oxo-dG distribution across the genome, a dissimilar context pattern to C>A mutations, and concurrent 5-mC depletion within a 2-kilobase window surrounding 8-oxo-dG sites. These findings not only underscore the potential of nanopore sequencing in epigenetic research, but also shed light on 8-oxo-dGs role in genomic regulation. By simultaneously measuring 5-mC and 8-oxo-dG at single molecule resolution, our study provides insights into the functional interplay between these DNA modifications. Moreover, our approach using synthetic oligos to generate a ground truth from machine learning modification calling could be applied to any other DNA modification. Overall, our work contributes to advancing the field of epigenetics and highlights nanopore sequencing as a powerful tool for studying DNA modifications.