How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.

人们对慢性突变过程和间断的 DNA 损伤如何驱动癌症基因组进化知之甚少。在这里，我们展示了一种策略，可以在单细胞有丝分裂期间和单链分辨率下解开和量化单细胞基因组进化的不同机制。为了区分慢性（活性氧（ROS））和急性（紫外线（UV））诱变，我们通过微流体将一对姐妹细胞从爆发性紫外线损伤后的第一次有丝分裂中分离出来。引人注目的是，紫外线突变表现为姐妹特异性事件，揭示了全基因组的镜像突变。相反，转录区域中的 ROS 突变会以不可知的方式减少链。在持续的紫外线损伤下，连续几轮的基因组复制驱动了 CC 二核苷酸的多等位基因变异。最后，我们证明突变定相可以解析为 F1 小鼠肝肿瘤整个基因组中的单链。该策略可广泛用于区分重叠的癌症相关突变过程的贡献。