Mutations create genetic variation for other evolutionary forces to operate on and cause numerous genetic diseases. Nevertheless, how de novo mutations arise remains poorly understood. Progress in the area is hindered by the fact that error rates of conventional sequencing technologies (1 in 100 or 1,000 base pairs) are several orders of magnitude higher than de novo mutation rates (1 in 10,000,000 or 100,000,000 base pairs per generation). Moreover, previous analyses of germline de novo mutations examined pedigrees (and not germ cells) and thus were likely affected by selection. Here, we applied highly accurate duplex sequencing to detect low-frequency, de novo mutations in mitochondrial DNA (mtDNA) directly from oocytes and from somatic tissues (brain and muscle) of 36 mice from two independent pedigrees. We found mtDNA mutation frequencies 2- to 3-fold higher in 10-month-old than in 1-month-old mice, demonstrating mutation accumulation during the period of only 9 mo. Mutation frequencies and patterns differed between germline and somatic tissues and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. Additionally, we discovered a more pronounced genetic drift of mitochondrial genetic variants in the germline of older versus younger mice, arguing for mtDNA turnover during oocyte meiotic arrest. Our study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing directly in oocytes, which provided unprecedented resolution and minimized selection effects present in pedigree studies. Moreover, our work provides important information about the origins and accumulation of mutations with aging/maturation and has implications for delayed reproduction in modern human societies. Furthermore, the duplex sequencing method we optimized for single cells opens avenues for investigating low-frequency mutations in other studies.

突变会产生遗传变异，使其他进化力量发挥作用并导致多种遗传疾病。然而，从头突变是如何产生的仍然知之甚少。该领域的进展受到以下事实的阻碍：传统测序技术的错误率（100或1000个碱基对中有1个）比从头突变率（每代10,000,000个或100,000,000个碱基对中有1个）高几个数量级。此外，之前对种系从头突变的分析检查的是谱系（而不是生殖细胞），因此可能受到选择的影响。在这里，我们应用高度准确的双链测序来直接检测来自两个独立谱系的 36 只小鼠的卵母细胞和体细胞组织（大脑和肌肉）的线粒体 DNA (mtDNA) 的低频从头突变。我们发现 10 个月大的小鼠的 mtDNA 突变频率比 1 个月大的小鼠高 2 至 3 倍，这表明突变在仅 9 个月的时间内就积累了。种系组织和体细胞组织之间以及 mtDNA 区域之间的突变频率和模式不同，表明存在不同的突变机制。此外，我们发现老年小鼠与年轻小鼠的种系中线粒体遗传变异的遗传漂移更为明显，这表明卵母细胞减数分裂停滞期间线粒体DNA发生了变化。我们的研究首次直接在卵母细胞中使用双链测序破译了种系从头诱变的复杂性，这提供了前所未有的分辨率并将谱系研究中存在的选择效应降至最低。此外，我们的工作提供了有关衰老/成熟过程中突变的起源和积累的重要信息，并对现代人类社会的延迟繁殖具有影响。 此外，我们针对单细胞优化的双链测序方法为其他研究中研究低频突变开辟了途径。