DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family.,Nature Protocols

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DNA copy-number measurement of genome replication dynamics by high-throughput sequencing: the sort-seq, sync-seq and MFA-seq family.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-02-12 , DOI: 10.1038/s41596-019-0287-7
Dzmitry G Batrakou ₁ , Carolin A Müller ₁ , Rosemary H C Wilson ₁ , Conrad A Nieduszynski ₁

Affiliation

Genome replication follows a defined temporal programme that can change during cellular differentiation and disease onset. DNA replication results in an increase in DNA copy number that can be measured by high-throughput sequencing. Here we present a protocol to determine genome replication dynamics using DNA copy-number measurements. Cell populations can be obtained in three variants of the method. First, sort-seq reveals the average replication dynamics across S phase in an unperturbed cell population; FACS is used to isolate replicating and non-replicating subpopulations from asynchronous cells. Second, sync-seq measures absolute replication time at specific points during S phase using a synchronized cell population. Third, marker frequency analysis can be used to reveal the average replication dynamics using copy-number analysis in any proliferating asynchronous cell culture. These approaches have been used to reveal genome replication dynamics in prokaryotes, archaea and a wide range of eukaryotes, including yeasts and mammalian cells. We have found this approach straightforward to apply to other organisms and highlight example studies from across the three domains of life. Here we present a Saccharomyces cerevisiae version of the protocol that can be performed in 7-10 d. It requires basic molecular and cellular biology skills, as well as a basic understanding of Unix and R.

中文翻译：

通过高通量测序对基因组复制动力学进行DNA拷贝数测量：sort-seq，sync-seq和MFA-seq家族。

基因组复制遵循定义的时间程序，该程序可以在细胞分化和疾病发作期间发生变化。DNA复制导致DNA拷贝数增加，可以通过高通量测序来测量。在这里，我们提出了使用DNA拷贝数测量来确定基因组复制动力学的协议。可以以该方法的三种变体获得细胞群体。首先，sort-seq揭示了不受干扰的细胞群中整个S期的平均复制动态；FACS用于从异步单元中隔离复制和非复制亚群。其次，sync-seq使用同步的细胞群来测量S阶段特定点的绝对复制时间。第三，标记频率分析可用于在任何增殖的异步细胞培养物中使用拷贝数分析揭示平均复制动力学。这些方法已用于揭示原核生物，古细菌和各种真核生物，包括酵母和哺乳动物细胞中的基因组复制动力学。我们发现该方法可直接应用于其他生物，并突出了生命三个领域的实例研究。在这里，我们介绍了可以在7-10天内执行的酿酒酵母版本的协议。它需要基本的分子和细胞生物学技能，以及对Unix和R的基本了解。包括酵母和哺乳动物细胞。我们发现该方法可直接应用于其他生物，并突出了生命三个领域的实例研究。在这里，我们介绍了可以在7-10天内执行的酿酒酵母版本的协议。它需要基本的分子和细胞生物学技能，以及对Unix和R的基本了解。包括酵母和哺乳动物细胞。我们发现该方法可直接应用于其他生物，并突出了生命三个领域的实例研究。在这里，我们介绍了可以在7-10天内执行的酿酒酵母版本的协议。它需要基本的分子和细胞生物学技能，以及对Unix和R的基本了解。

更新日期：2020-02-12

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