Massively Parallel Haplotyping

描述（由申请人提供）：由于人群分层和混合，遗传关联研究经常受到阳性和阴性混杂的影响。使用现代统计流行病学可以减轻阳性和阴性混杂，但无法解决。因此，许多遗传学家认为，遗传关联并没有实现它的承诺。因为遗传关联的主要目标是找到与遗传疾病相关的祖先单倍型，所以单倍型定相可以显著增加遗传关联的能力。我们提出了一种使用单个精子进行高通量单倍型分型的方法，这有可能彻底改变遗传关联研究。该方法来源于一系列技术（GigaLink），用于在数百万个单细胞中平行地分子共定位两个或更多个核酸靶标。概括地说，该技术将单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个单核苷酸多态性（SNP），然后通过下一代测序在反向乳液中对连锁基因座进行测序。这使得能够进行比如果仅分析单个细胞中的单个基因座或甚至跨许多单个细胞的单个基因座所可能进行的生物分析复杂得多的生物分析。在第一阶段，我们的目标是建立一个概念验证的方法，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优化，然后构建用于精子捕获的定制微流体装置。我们将通过比较单精子微阵列数据显示的相位与单精子GigaLink显示的相位来测试该方法的准确性。在第二阶段，我们将通过将重叠延伸引物组附着到珠上，然后每个微滴喷射一个珠，将该技术扩展到高度多重的精子单倍型。商业化的技术将彻底改变全基因组关联研究，最终提供可访问和准确的分阶段遗传数据。 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，将改善这些统计方法，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.