Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing
Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing
复制标题
使用冈崎片段测序对 DNA 复制叉方向性进行全基因组测量并定量 DNA 复制起始和终止
DOI：
10.1038/s41596-022-00793-5
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发表时间：
2023-01-18
期刊：
影响因子：
14.8
通讯作者：
Petryk，Nataliya
中科院分区：
文献类型：
--
作者：
Wu，Xia;Liu，Yaqun;Petryk，Nataliya
研究哺乳动物细胞中基因组复制的动力学一直具有历史挑战性。为了揭示人类基因组中复制起始和终止的位置，我们开发了Okazaki片段测序（OK-seq），这是一种基于滞后链复制中间体Okazaki片段的分离和链特异性测序的定量方法。OK-seq量化了每个基因组位点上向左和向右分叉的比例，揭示了复制起始和终止事件的位置和效率。在这里，我们提供了在未受干扰的培养的人类细胞和出芽酵母中进行OK-seq的详细实验程序，以及用于数据处理和复制叉方向性计算的生物信息学管道。此外，我们提出了基于隐马尔可夫模型的分析方法，该方法允许自动检测上升、下降和平坦的复制叉的方向性片段，揭示整个基因组中复制起始、终止和单向叉运动的区域。这些工具对于准确解释人类和酵母复制程序至关重要。实验和数据处理可在6天内完成。除了详细揭示基因组复制程序外，OK-seq还在许多研究中发挥了重要作用，揭示了基因组稳定性、表观基因组维持和基因组进化的机制。
Studying the dynamics of genome replication in mammalian cells has been historically challenging. To reveal the location of replication initiation and termination in the human genome, we developed Okazaki fragment sequencing (OK-seq), a quantitative approach based on the isolation and strand-specific sequencing of Okazaki fragments, the lagging strand replication intermediates. OK-seq quantitates the proportion of leftward- and rightward-oriented forks at every genomic locus and reveals the location and efficiency of replication initiation and termination events. Here we provide the detailed experimental procedures for performing OK-seq in unperturbed cultured human cells and budding yeast and the bioinformatics pipelines for data processing and computation of replication fork directionality. Furthermore, we present the analytical approach based on a hidden Markov model, which allows automated detection of ascending, descending and flat replication fork directionality segments revealing the zones of replication initiation, termination and unidirectional fork movement across the entire genome. These tools are essential for the accurate interpretation of human and yeast replication programs. The experiments and the data processing can be accomplished within six days. Besides revealing the genome replication program in fine detail, OK-seq has been instrumental in numerous studies unravelling mechanisms of genome stability, epigenome maintenance and genome evolution.