Streamlined cloning of auditory and vestibular mutants by whole genome sequencing

描述（由申请人提供）：在过去的二十年中，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由于在脊椎动物中使用前向基因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。现在已经进行了大量的遗传筛选，既针对一般的胚胎形态表型，也针对更专门的功能筛选，如平衡和听力。这些筛选在识别突变体方面非常成功，现在在斑马鱼中有超过6000种描述的化学诱导突变系。然而，由于目前定位克隆的困难，在这些细胞系中突变的基因只在不到一半的情况下被识别出来。目前位置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。新一代测序技术的最新进展已经将测序成本降低了几个数量级，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，但人们对斑马鱼和其他模式生物进行位置克隆的方式却几乎没有改变。本文以斑马鱼内耳突变体为研究对象，提出应用新一代测序技术简化模式生物突变体的定位克隆。我们的目标是将位置克隆的人工和成本降低一到两个数量级。我们将通过克隆10个现有的突变体——半规管形态发生和耳聋两类突变体，比较基于连锁和纯合子定位的两种不同的测序克隆方法。

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.