Streamlined cloning of auditory and vestibular mutants by whole genome sequencing

通过全基因组测序简化听觉和前庭突变体的克隆

基本信息

批准号：
8224539
负责人：
SEAN G MEGASON
金额：
$ 25.43万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-10 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8224539
关键词：
Adult Affect Alleles Animal Model Applied Genetics Area Auditory Base Sequence Bioinformatics Biological Chemicals Cloning Computer software DNA Resequencing Defect Development Ear Embryo Embryonic Development Equilibrium Future Gamma Rays Genes Genetic Genetic Polymorphism Genetic Screening Genome Genomics Goals Hair Health Hearing Histocompatibility Testing Human Human Development Individual Insertional Mutagenesis Investments Labyrinth Learning Maps Methods Modeling Molecular Molecular Analysis Morphogenesis Mutagenesis Mutagens Mutate Mutation Natural regeneration Organ Pathway interactions Phenotype Positioning Attribute Publishing Reaction Research Research Personnel Semicircular canal structure Structure Technology Time Vertebrates Zebrafish base cell type cellular transduction cost deafness feeding gene function genetic linkage analysis genome sequencing model organisms databases mutant next generation novel positional cloning prevent research study sound

项目摘要

DESCRIPTION (provided by applicant): Over the last two decades zebrafish has emerged as one of the preeminent model organisms. This rise was in large part due to the promise of using forward genetic screens in a vertebrate animal to discover and elucidate the function of genes relevant to human development and health. Numerous genetic screens have now been performed both for general embryonic morpohological phenotypes as well as more specialized functional screens such as for balance and hearing. These screens have been very successful in identifying mutants and there are now over 6000 described chemically induced mutant lines in zebrafish. However, the gene mutated in these lines has only been identified in less than half the cases due to the current difficulty of positional cloning. The high cost and labor currently required for positional cloning prevents a molecular analysis of many current mutants as well as discouraging future genetic screens. Recent advances in next generation sequencing have reduced the cost of sequencing by several orders of magnitude such that it is now possible to routinely resequence entire genomes. These advances have already revolutionized many areas of genomics yet the way people do positional cloning in zebrafish and other model organisms has changed relatively little. Here we propose to apply next generation sequencing technologies to streamline positional cloning of mutants in model organisms focusing on zebrafish inner ear mutants. Our goal is to reduce the labor and cost of positional cloning by one to two orders of magnitude. We will compare two different approaches for cloning-by-sequencing based on linkage and homozygosity mapping by cloning 10 existing mutants in two mutant classes-semicircular canal morphogenesis and deafness. PUBLIC HEALTH RELEVANCE: Defects in hearing and balance represent a major health problem in humans. Models for these defects can be identified in genetic screens in zebrafish but are currently difficult to molecularly characterize. We will develop an approach to streamline the cloning of such mutants using next generation sequencing.

描述（由申请人提供）：在过去的二十年中，斑马鱼已成为卓越的模式生物之一。这种增长在很大程度上是由于在脊椎动物中使用正向遗传筛选来发现和阐明与人类发育和健康相关的基因功能的前景。现在已经针对一般胚胎形态表型以及更专业的功能筛选（例如平衡和听力）进行了许多遗传筛选。这些筛选在识别突变体方面非常成功，目前已有 6000 多个斑马鱼化学诱导突变株系被描述。然而，由于目前定位克隆的困难，这些品系中的突变基因仅在不到一半的情况下被发现。目前定位克隆所需的高成本和劳动力阻碍了对许多当前突变体的分子分析，也阻碍了未来的遗传筛选。下一代测序的最新进展已将测序成本降低了几个数量级，因此现在可以对整个基因组进行常规重测序。这些进步已经彻底改变了基因组学的许多领域，但人们在斑马鱼和其他模式生物中进行定位克隆的方式变化相对较小。在这里，我们建议应用下一代测序技术来简化模型生物体中突变体的定位克隆，重点关注斑马鱼内耳突变体。我们的目标是将定位克隆的劳动力和成本减少一到两个数量级。我们将通过克隆两个突变体类别（半规管形态发生和耳聋）中的 10 个现有突变体，比较基于连锁和纯合性映射的两种不同的测序克隆方法。公共卫生相关性：听力和平衡缺陷是人类的一个主要健康问题。这些缺陷的模型可以通过斑马鱼的遗传筛选来鉴定，但目前很难进行分子表征。我们将开发一种使用下一代测序来简化此类突变体克隆的方法。