Massively Parallel Haplotyping

描述(由申请者提供)：由于群体中的分层和混合，遗传关联研究经常受到积极和消极的混淆。积极和消极的混淆可以缓解，但不能使用现代统计流行病学解决。因此，许多遗传学家提出，基因关联并没有兑现它的承诺。由于遗传关联的主要目标是寻找与遗传病相关的祖先单倍型，因此单倍型阶段化可以显著提高遗传关联的能力。我们提出了一种使用单个精子进行高通量单倍体鉴定的方法，这可能会给遗传关联研究带来革命性的变化。该方法源于一系列技术(GigaLink)，用于在数百万个单细胞中并行地分子共定位两个或更多个核酸目标。广泛地说，这项技术将单个细胞分离到油包水皮升反应器中，通过分子间杂交融合两个或更多单核苷酸多态(SNPs)，然后通过下一代测序在反向乳剂中对链接的基因座进行测序。这使得能够进行比仅分析单个细胞中的单个位置或甚至跨多个单个细胞的单个位置更复杂的生物学分析。在第一阶段，我们的目标是建立一种概念验证方法，用于单个精子捕获和两个核酸靶标之间的分子间连接。我们建议对重叠延伸引物进行彻底的优化，然后构建一种定制的微流控精子捕获装置。我们将通过将单精子微阵列数据揭示的相位与单精子GigaLink揭示的相位进行比较来测试该方法的准确性。在第二阶段，我们将把这项技术扩展到高度多元化的精子单倍化，方法是在珠子上贴上重叠延伸引物集，然后每个微滴喷射一个珠子。这项商业化的技术最终将提供可访问和准确的阶段性基因数据，从而使全基因组关联研究发生革命性变化。 公共卫生相关性：遗传学家研究大量无关个体，以了解常见疾病的遗传原因。这些研究依赖于容易出错的统计方法。我们正在开发一种新的分子方法，将改进这些统计方法，从而帮助遗传学家了解常见疾病的遗传原因。

DESCRIPTION (provided by applicant): Genetic association studies often suffer from positive and negative confounding due to stratification and admixture in populations. Positive and negative confounding can be alleviated but not solved using modern statistical epidemiology. As a result, many geneticists have suggested that genetic association has not lived up to its promise. Because the major goal of genetic association is to find ancestral haplotypes associated with genetic disease, haplotype phasing could significantly increase the power of genetic association. We propose a method for high throughput haplotyping using single sperm, which has the potential to revolutionize genetic association studies. The method is derived from a family of technologies (GigaLink) for molecular co-localization of two or more nucleic acid targets in millions of single cells in parallel. Broadly, the technology isolates single cells into aqueous-in-oil picoliter reactors, fuses two or more single nucleotide polymorphisms (SNPs) by intermolecular hybridization, and then sequences linked loci in reversed emulsions by next-generation sequencing. This enables far more complicated biological analysis than is possible if analyzing only a single locus in a single cell or even a single locus across many single cells. In Phase I, our objective is to build a proof-of-concept method for single sperm capture and intermolecular linkage between two nucleic acid targets. We propose to conduct a thorough optimization of overlap extension primers and then build a custom microfluidics device for sperm capture. We will test the accuracy of the method by comparing phase revealed by single sperm microarray data to phase revealed by single sperm GigaLink. In Phase II, we will extend the technology to highly multiplexed sperm haplotyping by affixing overlap extension primer sets to beads and then ejecting one bead per microdroplet. The commercialized technology will revolutionize genome-wide association studies by finally providing accessible and accurate phased genetic data. PUBLIC HEALTH RELEVANCE: Geneticists study large populations of unrelated individuals to understand the genetic cause of common diseases. These studies rely on error-prone statistical approaches. We are developing a new molecular method that will improve these statistical approaches and therefore help geneticists understand genetic causes of common disease.