Haplotype Resolved Sequencing Technology

单倍型解析测序技术

• 批准号: 8568163
• 负责人: Jeremy S Edwards
• 金额: $ 45万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8568163
• 关键词: Chromosomes; Cohort Studies; Commit; Communities; Complex; DNA Sequence; Data; Data Set; Development; Disease; Emulsions; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Genome; Genotype; Goals; Haplotypes; Hereditary Disease; Human; Human Genome; Libraries; Link; Methods; Patients; Phase; Phenotype; Play; Population; Preparation; Reading; Research Personnel; Role; System; Techniques; Technology; Testing; Variant; base; cost; design; genetic analysis; genetic variant; genome sequencing; human disease; improved; innovation; innovative technologies; neglect; next generation sequencing; open source; tool

DESCRIPTION (provided by applicant): Project Summary: The focus of this proposal is to develop an electrical/non-optical next generation sequencing technology to sequence a haplotype resolved human genome for less than $1,000. Recent advances in Next-Gen sequencing, along with the development of robust analysis methods, have given researchers the ability to determine the role of sequence variations in human diseases. However, the vast majority of sequencing technologies produce results that are limited to finding polymorphisms, and the importance of haplotypes has been largely neglected. In order to truly understand the genetic makeup of a specific disease there is a definite need to develop methods to identify the specific chromosome of all polymorphisms. This is because haplotypes are more effective for identifying disease-causing loci. To accomplish this goal we have defined three specific aims. Aim 1. We will develop a paired-end rolony sequencing strategy. Rolonies are rolling circle amplicon colonies. Rolonies are a very promising replacement for emulsion PCR beads. Rolonies are attractive due to their simplicity and small size. However, paired-end rolony sequencing is currently not possible using sequencing-by-synthesis, and we strongly believe paired-end reads are essential to allow for complete haplotype resolved assembly of a human genome. Therefore, aim 1 is devoted to establishing the paired-end sequencing approach. Our paired-end rolony sequencing strategy will be developed and tested using reversible terminators that are conjugated to a cleavable fluorescent moiety. We will then apply and optimize the paired-end sequencing strategy in our ISFET electrical sequencing device using reversible terminators without fluorescent labels. Aim 2. We will develop a sequencing strategy that allows for assembly of the human genome while maintaining haplotype information. We have termed our approach haplotype resolved whole genome sequencing (hrWGS). We believe that for human genome sequence information to be most valuable, haplotypes must be identified. Haplotypes are important for identifying the association between genetic variations and disease. Furthermore, haplotype information is a key factor in the genetic analysis of populations. Currently, not a single next- generation sequencing technology is designed to deliver haplotype information. We propose to develop two strategies for haplotype resolved sequencing. Aim 3. We will develop a non-light based sequencing strategy for paired-end rolonies. The non-light based strategy will employ a high density ISFET array to detect the biochemical events that occur when a base is incorporated during sequencing-by-synthesis. Our non-light based sequencing strategy offers tremendous improvements over existing technologies. Namely, we will combine rolony sequencing with innovative ISFET designs to allow for 10B sensors per chip. Additionally, we will utilize our innovative paired-end sequencing strategy and novel library preparation methods to allow for haplotype resolution. In conclusion, our innovative technology provides significant advances beyond currently available approaches and will significantly advance the current state-of-the-art. We estimate that we could sequence a haplotype resolved human genome for much less than $1,000 using the proposed technology.

描述（由申请人提供）：项目摘要：该提案的重点是开发一种电气/非光学下一代测序技术，以将单倍型解析的人类基因组对人类基因组进行少于1,000美元。下一代测序的最新进展以及强大的分析方法的发展使研究人员能够确定序列变化在人类疾病中的作用。但是，绝大多数测序技术产生的结果仅限于寻找多态性，而单倍型的重要性在很大程度上被忽略了。为了真正了解特定疾病的基因组成，确定需要开发方法来识别所有多态性的特定染色体。这是因为单倍型对于鉴定引起疾病的基因座更有效。为了实现这一目标，我们定义了三个特定目标。 AIM 1。我们将制定配对的Rolony测序策略。 rolonies是滚动圆的扩增子殖民地。 Rolonies是乳液PCR珠的一个非常有希望的替代品。 Rolonies由于其简单性和尺寸很小而具有吸引力。然而，目前不可能使用逐个测序来进行配对的rolony测序，我们坚信，配对末端读数对于允许人类基因组的完整单倍型分解组装至关重要。因此，AIM 1致力于建立配对的测序方法。我们的配对末端rolony测序策略将使用可逆终结器制定和测试，这些终止剂与可裂解的荧光部分结合在一起。然后，我们将使用无荧光标签的可逆终结器在我们的ISFET电序列设备中应用并优化配对端的测序策略。 AIM 2。我们将制定一种测序策略，该策略允许在维持单倍型信息的同时组装人类基因组。我们称我们的方法单倍型解决了整个基因组测序（HRWGS）。我们认为，要使人类基因组序列信息最有价值，必须确定单倍型。单倍型对于确定遗传变异与疾病之间的关联很重要。此外，单倍型信息是人群遗传分析的关键因素。目前，没有一种下一代测序技术旨在提供单倍型信息。我们建议制定两种单倍型解决测序的策略。 AIM 3。我们将针对配对的rolonies制定基于非光线的测序策略。基于非光的策略将采用高密度ISFET阵列来检测在测序过程中掺入基础时发生的生化事件。我们基于非灯的测序策略对现有技术提供了巨大的改进。也就是说，我们将将Rolony测序与创新的ISFET设计相结合，以每个芯片允许10B传感器。此外，我们将利用我们创新的配对端测序策略和新图书馆 制备方法允许单倍型分辨率。 总而言之，我们的创新技术在当前可用的方法之外还提供了重大进步，并将大大推动当前的最新方法。我们估计，使用拟议的技术，我们可以以不到1,000美元的价格对单倍型的解决人类基因组进行测序。