Massively Parallel Haplotyping

大规模平行单倍型分析

• 批准号: 8198444
• 负责人: David Scott Johnson
• 金额: $ 18.96万
• 依托单位: GIGAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8198444
• 关键词: Admixture; Biological; Buffers; Cells; Complex; Custom; Cytolysis; DNA; Data; Disease; Emulsions; Ensure; Epidemiology; Family; Genetic; Goals; Haplotypes; Hereditary Disease; Human; Individual; Libraries; Life; Link; Methods; Microfluidic Microchips; Microfluidics; Molecular; Nucleic Acids; Oils; Phase; Polymerase Chain Reaction; Population; Primer Extension; Reaction; Recovery; Single Nucleotide Polymorphism; Solutions; Stratification; System; Technology; Testing; Validation; Variant; aqueous; base; design; genetic association; genetic variant; genome wide association study; improved; next generation; sperm cell

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.

描述（由申请人提供）：遗传关联研究通常由于人口分层和混合而遭受正面和负面混杂。可以通过现代统计流行病学来缓解积极和消极的混杂，但不能解决。结果，许多遗传学家提出遗传关联并未达到其承诺。由于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，因此单倍型相位可以显着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吞吐量单倍型的方法，该方法有可能彻底改变遗传关联研究。该方法源自一系列技术（GIGALINK），用于平行数百万个单个细胞中两个或多个核酸靶标的分子共定位。从广义上讲，该技术将单个细胞分离到油中的picoliter反应器中，通过分子间杂交融合了两个或多个单一核苷酸多态性（SNP），然后通过下一代测序将反向的乳液中的序列连接到反向乳液中。如果仅分析单个细胞中的一个基因座，甚至在许多单个单元中仅分析单个基因座，则可以使生物学分析更复杂。在第一阶段，我们的目标是构建一种概念验证方法，用于两个核酸靶标之间的单个精子捕获和分子间连接。我们建议对重叠扩展引物进行彻底优化，然后构建一个自定义的微流体设备以捕获精子。我们将通过比较单个精子微阵列数据与单个精子GigAlink揭示的相位的相位来测试该方法的准确性。在II阶段，我们将通过将重叠式伸展引物集固定到珠子，然后每个微光谱弹出一个珠子，将技术扩展到高度多路复用的精子单倍型。商业化技术将通过最终提供可访问且准确的分阶段遗传数据来彻底改变全基因组关联研究。 公共卫生相关性：遗传学家研究大量无关的个体人群，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，该方法将改善这些统计方法，因此有助于遗传学家了解常见疾病的遗传原因。