Mutant mapping and identification in zebrafish by next generation sequencing

通过下一代测序对斑马鱼进行突变定位和鉴定

• 批准号: 8334932
• 负责人: DAVID R. BEIER
• 金额: $ 53.04万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8334932
• 关键词: Base Sequence; Biological Models; Biological Process; Chromosome Mapping; Communities; Complement; Computational Technique; Computer Simulation; Computer software; Computing Methodologies; DNA; DNA Sequence; Data; Databases; Development; Developmental Biology; Experimental Designs; Expressed Sequence Tags; Fishes; Gene Mutation; Genes; Genetic; Genetic Markers; Genetic Recombination; Genetic Variation; Genomics; Investigation; Maps; Methods; Microsatellite Repeats; Modeling; Mutagenesis; Mutation; Phenotype; Population; Positioning Attribute; Protocols documentation; Relative (related person); Sampling; Scanning; Siblings; Software Tools; Source; Statistical Methods; System; Technology; Variant; Zebrafish; base; comparative genomics; computerized tools; cost effective; design; experimental analysis; gene discovery; gene function; genetic analysis; genetic manipulation; genome sequencing; human disease; knowledge translation; mutant; next generation; positional cloning; rapid technique; research study; stem; web-accessible; zebrafish genome

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.

描述(由申请人提供)：斑马鱼系统的力量来自于它作为发育生物学模型的实用性，以及它的遗传操作和实验的简便性。斑马鱼诱导和自发突变的表型特征、遗传图谱和位置克隆推动了我们对脊椎动物发育关键遗传机制的理解。然而，该系统的潜力还没有完全实现，因为效率低下的基于微卫星的测绘仍然是该领域的主要方法。我们建议将现代基因组学的技术和计算进展应用到斑马鱼系统的遗传图谱中。具体地说，我们建议开发一种使用混合样本的下一代测序(NGS)来快速和准确地定位隐性斑马鱼突变体的方法。我们还建议研究屏幕设计和样本分析的参数，以优化该协议的使用。最后，我们的目标是开发在图谱区间内发现的变异中识别因果突变的方法。NGS技术的应用，辅以专门开发的计算技术，将为斑马鱼的遗传图谱提供一种高效、准确和廉价的方法。这种方法将能够在单个实验中同时识别信息丰富的遗传标记、突变位置的图谱以及潜在的因果序列变化的识别。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界。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： 对斑马鱼突变体的分析使人们能够识别与包括人类疾病在内的基本生物学过程有关的基因；然而，用于突变定位和基因发现的方法效率低下。在这里，我们提出了一种使用下一代测序进行遗传作图和突变识别的快速且经济有效的方法。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。