Streamlined cloning of auditory and vestibular mutants by whole genome sequencing

描述(申请人提供)：在过去的二十年里，斑马鱼已经成为卓越的模式生物之一。这一增长在很大程度上是因为有望在脊椎动物中使用正向基因筛查来发现和阐明与人类发育和健康相关的基因的功能。现在已经进行了许多遗传筛查，既针对一般的胚胎形态表型，也针对更专门的功能筛查，如平衡和听力。这些筛选在鉴定突变方面非常成功，目前已有6000多个斑马鱼化学诱导突变系被描述。然而，由于目前位置克隆的困难，这些品系中突变的基因只在不到一半的病例中被识别出来。目前位置克隆所需的高昂成本和劳动力阻碍了对许多当前突变的分子分析，并阻碍了未来的基因筛选。新一代测序技术的最新进展将测序成本降低了几个数量级，因此现在有可能对整个基因组进行常规的重新测序。这些进展已经给基因组学的许多领域带来了革命性的变化，但人们在斑马鱼和其他模式生物中进行位置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式生物中突变体的位置克隆，重点是斑马鱼内耳突变体。我们的目标是将位置克隆的劳动力和成本降低一到两个数量级。我们将通过克隆两个突变类别的10个现有突变-半规管形态发生和耳聋，比较两种不同的基于连锁和纯合作图的克隆方法。

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.