Pagès-Gallego Marc, van Soest Daan M K, Besselink Nicolle J M, Straver Roy, Keijer Janneke P, Vermeulen Carlo, Marcozzi Alessio, van Roosmalen Markus J, van Boxtel Ruben, Burgering Boudewijn M T, Dansen Tobias B, de Ridder Jeroen
Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Oncode Institute, Utrecht, The Netherlands.
Nat Commun. 2025 Jun 5;16(1):5236. doi: 10.1038/s41467-025-60391-3.
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.
基因组DNA不断受到氧化损伤，8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧代-dG）是与诱变、表观遗传学和基因调控相关的主要损伤。现有的检测8-氧代-dG的方法依赖于间接方法，而纳米孔测序能够直接检测碱基修饰。由于缺乏训练数据，目前还没有8-氧代-dG检测模型。在这里，我们开发了一种策略，使用合成寡核苷酸来生成用于深度学习和纳米孔测序的长的、8-氧代-dG上下文可变的DNA分子。我们的训练方法解决了8-氧代-dG相对于鸟嘌呤的稀有性问题，能够实现特异性检测。应用于氧化损伤的组织培养模型，我们的方法揭示了基因组8-氧代-dG分布不均匀、与C>A突变不同的上下文模式以及局部5-甲基胞嘧啶（5-mC）消耗。这种在单分子分辨率下对5-mC和8-氧代-dG的双重测量揭示了它们相互作用的新见解。我们的方法还为使用合成DNA和纳米孔测序检测其他罕见的DNA修饰提供了一个通用框架。
