David Serena S, Pacheco Brendan A, Kishimoto Kensei, Vantine Sam, Hu Kai, Liu Haibo, Davis Diana L, Tran Hoang, Sallis Benjamin F, Ali Levi, Haynes Cole M, McCormick Beth A, Zhu Lihua Julie, Flavahan William A
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Department of Microbiology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Nat Commun. 2025 Jul 1;16(1):5676. doi: 10.1038/s41467-025-60896-x.
DNA exists biologically as a highly dynamic macromolecular complex subject to myriad chemical modifications that alter its physiological interpretation, yet most sequencing technologies only measure Watson-Crick base pairing interactions. Third-generation sequencing technologies can directly detect novel and modified bases, yet the difficulty and cost of training these techniques for each novel base has so far limited this potential. Here, we present a method based on barcoded split-pool synthesis to generate reference standard oligonucleotides allowing novel base sequencing. Using novel base detection, we perform multidimensional sequencing to retrieve information, both physiologically stored and experimentally encoded, from DNA, allowing us to characterize the preferential replication of deleterious mitochondrial genome mutations, the infection dynamics of a host-pathogen model, and the effect of chemotherapy on cancer cell DNA at the single molecule level. The low cost and experimental simplicity of this method make this approach widely accessible to the research community, enabling complex experimental interrogation across the biological sciences.
DNA在生物学上以一种高度动态的大分子复合物形式存在，会受到无数化学修饰的影响，这些修饰会改变其生理学解释，但大多数测序技术仅测量沃森-克里克碱基配对相互作用。第三代测序技术可以直接检测新的和修饰的碱基，但到目前为止，针对每个新碱基训练这些技术的难度和成本限制了这种潜力。在这里，我们提出了一种基于条形码拆分池合成的方法，以生成允许新碱基测序的参考标准寡核苷酸。通过新碱基检测，我们进行多维测序以从DNA中检索生理存储和实验编码的信息，从而使我们能够在单分子水平上表征有害线粒体基因组突变的优先复制、宿主-病原体模型的感染动态以及化疗对癌细胞DNA的影响。该方法的低成本和实验简单性使研究界能够广泛使用这种方法，从而能够在整个生物科学领域进行复杂的实验研究。
