Direct detection of 8-oxo-dG using nanopore sequencing
Marc Pagès-Gallego, Daan M. K. van Soest, Nicolle Besselink, Roy Straver, Janneke P. Keijer, Carlo Vermeulen, Alessio Marcozzi, Markus J. van Roosmalen, Ruben van Boxtel, Boudewijn Burgering, Tobias B. Dansen, Jeroen de Ridder
Abstract
Genomic DNA is under constant oxidative damage, with 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) being the prominent lesion linked to mutagenesis, epigenetics, and gene regulation. Existing methods to detect 8-oxo-dG rely on indirect approaches, while nanopore sequencing enables direct detection of base modifications. A model for 8-oxo-dG detection is currently missing due to the lack of training data. Here, we develop a strategy using synthetic oligos to generate long, 8-oxo-dG context-variable DNA molecules for deep learning and nanopore sequencing. Our training approach addresses the rarity of 8-oxo-dG relative to guanine, enabling specific detection. Applied to a tissue culture model of oxidative damage, our method reveals uneven genomic 8-oxo-dG distribution, dissimilar context pattern to C>A mutations, and local 5-mC depletion. This dual measurement of 5-mC and 8-oxo-dG at single-molecule resolution uncovers new insights into their interplay. Our approach also provides a general framework for detecting other rare DNA modifications using synthetic DNA and nanopore sequencing. Oxidative DNA damage is linked to cancer and aging, but its genomic effects remain hard to map. Here, authors develop a nanopore-based method using synthetic DNA and deep learning to detect 8- oxo-dG, revealing its distribution and interplay with DNA methylation.