High throughput methods for Synthetic Genetic Array Analysis in C. elegans

描述（由申请人提供）： 在线虫C.秀丽隐杆线虫已经适应了传统的遗传学研究方法。在酵母双突变体的系统研究中，利用酵母缺失收集，揭示了基因组中基因之间的相互作用，并允许推断大多数功能未知的基因的功能。在这里，我们建议开发的方法，将允许类似的研究在C。优雅我们将利用最近完成的测序突变株资源，这些突变株包含基因组中几乎每个基因的非同义变化。将这些菌株暴露于RNAi以寻找相互作用，如与针对野生型和其他突变菌株的RNAi或不存在RNAi的菌株相比的差异所反映的。为了提高效率和简化比较，菌株将在100个或更多的池中相互竞争，使用生长作为许多表型的替代。将使用分子倒置探针（MIP）跟踪合并液中单个菌株的丰度，以获得通过高通量测序定量的独特突变。通过比较相对于对照、其他RNA和群体遗传模型的丰度，我们将确定哪些菌株与每种RNAi表现出显著的正相互作用或负相互作用。为了确定每个菌株内的各种突变中的哪一种是与RNAi相互作用的基础，我们将每个相互作用菌株与多重标记的菌株杂交，在池中与RNAi竞争后代，并使用批量分离分析来鉴定相互作用的基因座。我们将使用来自包括来自文献的阳性和阴性对照的基因、来自作为二聚体但单独地被抑制的钾通道的twk家族的基因、以及来自广泛代表基因组的基因的一组RNAi来测试所述方法，以评估所述方法的总体效率。这些方法的成功实施将为系统地研究整个蠕虫基因组中的SGA铺平道路，为以前不知道功能的基因的作用提供有价值的见解。它还将为更详细地研究这些基因和网络提供一个框架。它也可能启发开发更复杂的生物体的类似方法。更全面地了解这种模式后生动物中的遗传相互作用，反过来可以深入了解人类中可能的基因-基因相互作用。

DESCRIPTION (provided by applicant): Only a fraction of the more than 20,000 genes in the nematode C. elegans have been amenable to traditional methods of genetic study. In yeast systematic investigations of double mutants, exploiting the yeast deletion collection, have revealed interactions between genes across the genome and have permitted inference of function for most genes of otherwise unknown function. Here we propose to develop methods that will permit analogous studies in C. elegans. We will exploit a recently completed resource of sequenced mutant strains that contains nonsynonymous changes in almost every gene in the genome. These strains will be exposed to RNAi to look for interactions as reflected in differences compared to the RNAi against wild type and other mutant strains or the strain in the absence of RNAi. To increase the efficiency and simplify comparison, the strains will be competed against one another in pools of 100 or more, using growth as a surrogate for a host of phenotypes. The abundance of individual strains in the pools will be followed using molecular inversion probes (MIPs) for the unique mutations quantified by high-throughput sequencing. By comparing the abundance relative to controls, other RNAs and population genetic models, we will determine which strains show a significant positive or negative interaction with each RNAi. To determine which of the various mutations within each strain underlie the interaction with the RNAi, we will cross each interacting strain with a multiply marked strain, compete the progeny in a pool with the RNAi and use bulk segregant analysis to identify the interacting loci. We will test the methods using a battery of RNAi's from genes that include positive and negative controls from the literature, from genes of the twk- family of potassium channels that act as dimers but are individually dispensable, and from genes broadly representative of the genome to assess the overall efficiency of the methods. The successful implementation of these methods will pave the way for systematic investigation of SGA across the worm genome, providing valuable insight into the role of genes without previously known function. It will also provide a framework for more detailed investigations of these genes and networks. It may also inspire the development of analogous methods for still more complex organisms. A more comprehensive understanding of genetic interactions in this model metazoan can in turn provide insight into likely gene-gene interactions in human.