Haplotype Resolved Sequencing Technology

单倍型解析测序技术

• 批准号: 8728983
• 负责人: Jeremy S Edwards
• 金额: $ 44.1万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 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/8728983
• 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/非光学下一代测序技术，以不到 1,000 美元的价格对单倍型解析的人类基因组进行测序。下一代测序的最新进展以及稳健分析方法的发展使研究人员能够确定序列变异在人类疾病中的作用。然而，绝大多数测序技术产生的结果仅限于发现多态性，而单倍型的重要性在很大程度上被忽视了。为了真正了解特定疾病的基因组成，确实需要开发识别所有多态性的特定染色体的方法。这是因为单倍型对于识别致病位点更有效。为了实现这一目标，我们确定了三个具体目标。 目标 1. 我们将开发双端 rolony 测序策略。 Rolonies 是滚环扩增子集落。 Rolonies 是乳液 PCR 微珠非常有前途的替代品。 Rolonies 因其简单和小尺寸而具有吸引力。然而，目前无法使用合成测序来实现双端 rolony 测序，我们坚信双端读取对于实现人类基因组的完整单倍型解析组装至关重要。因此，目标1致力于建立双端测序方法。我们的双端 rolony 测序策略将使用与可裂解荧光部分缀合的可逆终止子进行开发和测试。然后，我们将使用不带荧光标记的可逆终止子，在我们的 ISFET 电测序装置中应用和优化双端测序策略。 目标 2：我们将开发一种测序策略，允许在保留单倍型信息的同时组装人类基因组。我们将我们的方法称为单倍型解析全基因组测序（hrWGS）。我们相信，为了使人类基因组序列信息最有价值，必须鉴定单倍型。单倍型对于识别遗传变异与疾病之间的关联非常重要。此外，单倍型信息是群体遗传分析的关键因素。目前，还没有一种下一代测序技术被设计用来提供单倍型信息。我们建议开发两种单倍型解析测序策略。 目标 3. 我们将为双端 rolonies 开发一种基于非光的测序策略。基于非光的策略将采用高密度 ISFET 阵列来检测合成测序期间掺入碱基时发生的生化事件。我们的非光测序策略比现有技术提供了巨大的改进。也就是说，我们将把 rolony 测序与创新的 ISFET 设计相结合，以允许每个芯片支持 10B 传感器。此外，我们将利用我们创新的双端测序策略和新颖的文库 允许单倍型解析的制备方法。 总之，我们的创新技术提供了超越当前可用方法的重大进步，并将显着推进当前的最先进技术。我们估计，使用所提出的技术，我们可以以远低于 1,000 美元的价格对解析人类基因组的单倍型进行测序。