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τ-SGA: synthetic genetic array analysis for systematically screening and quantifying trigenic interactions in yeast
Nature Protocols ( IF 13.1 ) Pub Date : 2021-01-18 , DOI: 10.1038/s41596-020-00456-3
Elena Kuzmin 1, 2, 3 , Mahfuzur Rahman 4 , Benjamin VanderSluis 4 , Michael Costanzo 1 , Chad L Myers 4 , Brenda J Andrews 1, 2 , Charles Boone 1, 2
Affiliation  

Systematic complex genetic interaction studies have provided insight into high-order functional redundancies and genetic network wiring of the cell. Here, we describe a method for screening and quantifying trigenic interactions from ordered arrays of yeast strains grown on agar plates as individual colonies. The protocol instructs users on the trigenic synthetic genetic array analysis technique, τ-SGA, for high-throughput screens. The steps describe construction of the double-mutant query strains and the corresponding single-mutant control query strains, which are screened in parallel in two replicates. The screening experimental set-up consists of sequential replica-pinning steps that enable automated mating, meiotic recombination and successive haploid selection steps for the generation of triple mutants, which are scored for colony size as a proxy for fitness, which enables the calculation of trigenic interactions. The procedure described here was used to conduct 422 trigenic interaction screens, which generated ~460,000 yeast triple mutants for trigenic interaction analysis. Users should be familiar with robotic equipment required for high-throughput genetic interaction screens and be proficient at the command line to execute the scoring pipeline. Large-scale screen computational analysis is achieved by using MATLAB pipelines that score raw colony size data to produce τ-SGA interaction scores. Additional recommendations are included for optimizing experimental design and analysis of smaller-scale trigenic interaction screens by using a web-based analysis system, SGAtools. This protocol provides a resource for those who would like to gain a deeper, more practical understanding of trigenic interaction screening and quantification methodology.



中文翻译:

τ-SGA:用于系统筛选和量化酵母中三基因相互作用的合成遗传阵列分析

系统的复杂遗传相互作用研究提供了对细胞的高阶功能冗余和遗传网络布线的深入了解。在这里,我们描述了一种方法,用于筛选和量化从在琼脂板上作为单个菌落生长的有序酵母菌株阵列的三基因相互作用。该协议指导用户使用三基因合成遗传阵列分析技术,τ-SGA,用于高通量屏幕。这些步骤描述了双突变体查询菌株和相应的单突变体对照查询菌株的构建,它们在两个重复中并行筛选。筛选实验设置包括连续的复制固定步骤,这些步骤可以实现自动交配、减数分裂重组和连续的单倍体选择步骤,以生成三重突变体,根据菌落大小对它们进行评分,作为适应度的代表,从而能够计算三基因相互作用。此处描述的程序用于进行 422 个三基因相互作用屏幕,生成 ~ 460,000 个酵母三重突变体用于三基因相互作用分析。用户应熟悉高通量基因交互屏幕所需的机器人设备,并精通命令行来执行评分管道。大规模屏幕计算分析是通过使用 MATLAB 管道实现的,该管道对原始菌落大小数据进行评分以生成 τ-SGA 交互分数。还包括其他建议,用于通过使用基于网络的分析系统 SGAtools 优化小规模三基因交互屏幕的实验设计和分析。

更新日期:2021-01-18
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