Genome-wide mapping of individual replication fork velocities using nanopore sequencing. undefined

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed (NFS), a nanopore sequencing-based method to map and extract the velocity of individual replication forks detected as tracks of the thymidine analog bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NFS accurately retrieves previous genome-wide, mean speed estimates in Saccharomyces cerevisiae and precisely quantifies speed changes in cells exposed to hydroxyurea and in mutants with altered replisome progression. NFS surpasses standard approaches in simplicity, rapidity, throughput, spatial and temporal resolutions. Furthermore, the positioning of >125,000 single fork velocities provides the first-ever genome-wide map of fork progression based on the direct measurement of individual replication fork rates, showing a largely uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites. BO to BY samples in the training and testing dataset correspond to various mixtures of BrdU and thymidine (0:100; 10:90; 20:80; 30:70; 40:60; 50:50; 60:40; 70:30; 80:20; 90:10 and 100:0).

目前学界对真核生物基因组内复制叉速度的变异规律尚缺乏充分认知，因为现有测定复制叉速度的经典技术要么无法提供序列信息，要么存在通量低下的缺陷。本研究提出了NanoForkSpeed（NFS）：一种基于纳米孔测序的方法，可对单个复制叉的速度进行定位与提取；该技术通过对短暂脉冲标记异步增殖细胞时掺入的胸苷类似物溴脱氧尿苷（bromodeoxyuridine, BrdU）所形成的信号轨迹进行检测，实现单个复制叉的识别。该方法可准确复现酿酒酵母（Saccharomyces cerevisiae）全基因组范围内的平均复制速度既往估算结果，并能精准量化羟基脲（hydroxyurea）处理细胞以及复制体（replisome）进程异常的突变株中的复制速度变化。相较于传统标准方法，NFS在操作简便性、实验快速性、测序通量以及时空分辨率上均具有显著优势。此外，本研究对超过125,000个单个复制叉的速度进行了定位与量化，由此得到了首个基于单个复制叉速率直接测量的全基因组复制叉进程图谱。分析结果显示，酿酒酵母各染色体上的复制速度整体较为均一，仅在已知的复制暂停位点存在显著的速度下降。训练与测试数据集内的BO至BY样本，对应溴脱氧尿苷与胸苷的一系列混合比例（0:100、10:90、20:80、30:70、40:60、50:50、60:40、70:30、80:20、90:10及100:0）。