Mutant mapping and identification in zebrafish by next generation sequencing

描述（由申请人提供）：斑马鱼系统的力量源于其作为发育生物学模型的实用性及其遗传操作和实验的便利性。我们对脊椎动物发育的关键遗传机制的理解已经被斑马鱼诱导和自发突变的表型表征、遗传图谱和定位克隆所推动。然而，这一系统的潜力尚未得到充分发挥，因为基于微型卫星的测绘仍然是该领域的主要方法，效率低下。我们建议将当今基因组学的技术和计算进步应用于斑马鱼系统的遗传作图。具体而言，我们建议开发一种方法，使用下一代测序（NGS）的合并样本的隐性斑马鱼突变体的快速和准确的映射。我们还建议研究筛选设计和样品分析的参数，以优化该协议的使用。最后，我们的目标是开发用于识别映射间隔内发现的变体之间的因果突变的方法。应用NGS技术，辅以专门开发的计算技术，将提供一个有效的，准确的和廉价的方法在斑马鱼的遗传作图。这种方法将使信息的遗传标记的同时识别，突变位置的映射，并在一个单一的实验中的因果序列变化的潜在识别。在这些基因组分析中获得的数据和开发的方法将提供给斑马鱼社区。重要的是，这些方法也将广泛适用于其他模型系统的遗传分析。 公共卫生相关性： 斑马鱼突变体的分析已经能够识别基因有助于基本的生物过程，包括人类疾病，然而，用于突变体定位和基因发现的方法是低效的。 在这里，我们提出了一个快速和具有成本效益的方法，遗传图谱和突变识别使用下一代测序。 这将有助于更快地发现基因功能，并将这些知识转化为生物医学研究。

DESCRIPTION (provided by applicant): The power of the zebrafish system stems from its utility as a developmental biology model combined with the ease of its genetic manipulation and experimentation. Our understanding of key genetic mechanisms of vertebrate development has been propelled by the phenotypic characterization, genetic mapping and positional cloning of induced and spontaneous mutations in zebrafish. However, the potential of this system has not been fully realized, as inefficient microsatellite-based mapping remains the primary method in the field. We propose to apply technological and computational advances of present day genomics to genetic mapping in the zebrafish system. Specifically, we propose to develop a method for rapid and accurate mapping of recessive zebrafish mutants using Next Generation Sequencing (NGS) of pooled samples. We also propose to investigate parameters of screen design and sample analysis to optimize the use of this protocol. Finally, we aim to develop methods for identification of the causal mutation among the variants discovered within the mapping interval. Application of NGS technology, complemented by specifically developed computational techniques, will provide an efficient, accurate and inexpensive method for genetic mapping in zebrafish. This approach will enable the simultaneous identification of informative genetic markers, mapping of the mutation position, and potential identification of the causal sequence change in a single experiment. The data obtained in these genomic analyses and the methods developed will be made available to the zebrafish community. Importantly, these approaches will also be widely applicable to genetic analysis of other model systems. PUBLIC HEALTH RELEVANCE: Analysis of zebrafish mutants has enabled the identification of genes contributing to fundamental biological processes, including human diseases; however, the methods used for mutant mapping and gene discovery are inefficient. Here, we propose a fast and cost-effective method for genetic mapping and mutation identification using Next Generation Sequencing. This will facilitate the more rapid discovery of gene function and the translation of this knowledge to biomedical investigation.