Epigenetics and Cell Fate, CNRS, Université de Paris, Paris, France.
Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, CNRS, INSERM, Université PSL, Paris, France.
Methods Mol Biol. 2022;2477:107-128. doi: 10.1007/978-1-0716-2257-5_8.
Most genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200 nucleotide resolution using a nanopore current interpretation tool allowing the quantification of BrdU incorporation. Along pulse-chased replication intermediates from Saccharomyces cerevisiae, we can orient replication tracks and reproduce population-based replication directionality profiles. Additionally, we can map individual initiation and termination events. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.
大多数基因组复制图谱绘制方法对细胞群体进行分析，掩盖了细胞间的异质性。在这里，我们描述了 FORK-seq，这是一种纳米孔测序方法，可使用纳米孔电流解释工具以 200 个核苷酸的分辨率绘制单个 DNA 分子的复制图谱，该工具允许定量 BrdU 掺入。在来自酿酒酵母的脉冲追踪复制中间体中，我们可以定向复制轨迹并再现基于群体的复制方向性图谱。此外，我们还可以绘制单个起始和终止事件。因此，FORK-seq 揭示了 DNA 复制中细胞间异质性的全貌。
