Altmann Melina, Altmann Stefan, Falter Claudia, Falter-Braun Pascal
Helmholtz Zentrum München, Institute of Network Biology (INET), Munich, Germany.
Ludwig-Maximilians-Universität München, Microbe-Host Interactions, Munich, Germany.
Curr Protoc Plant Biol. 2018 Sep;3(3):e20067. doi: 10.1002/cppb.20067. Epub 2018 Jun 26.
In this article, we describe a Y2H interaction mapping protocol for systematically screening medium-to-large collections of open reading frames (ORFs) against each other. The protocol is well suited for analysis of focused networks, for modules of interest, assembling genome-scale interactome maps, and investigating host-microbe interactions. The four-step high-throughput protocol has a demonstrated low false-discovery rate that is essential for producing meaningful network maps. Following the assembly of yeast expression clones from an existing ORFeome collection, we describe in detail the four-step procedure that begins with the primary screen using minipools, followed by secondary verification of primary positives, identification of candidate interaction pairs by sequencing, and a final verification step using fresh inoculants. In addition to this core protocol, aspects of network mapping and quality control will be discussed briefly. © 2018 by John Wiley & Sons, Inc.
在本文中，我们描述了一种酵母双杂交相互作用图谱绘制方案，用于系统地相互筛选中等至大型的开放阅读框（ORF）集合。该方案非常适合分析聚焦网络、感兴趣的模块、组装基因组规模的相互作用组图谱以及研究宿主-微生物相互作用。这个四步高通量方案具有已证实的低错误发现率，这对于生成有意义的网络图谱至关重要。从现有的ORFeome集合组装酵母表达克隆后，我们详细描述了这个四步程序，该程序始于使用微量池进行初步筛选，接着对初步阳性结果进行二次验证，通过测序鉴定候选相互作用对，以及使用新鲜接种物进行最终验证步骤。除了这个核心方案外，还将简要讨论网络图谱绘制和质量控制方面的内容。© 2018约翰威立父子公司。
