Haplotype Resolved Sequencing Technology

描述（由申请人提供）：项目概述：本提案的重点是开发一种电/非光学下一代测序技术，以不到1000美元的价格对单倍型分辨的人类基因组进行测序。下一代测序的最新进展，沿着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遗传组成，确实需要开发鉴定所有多态性的特定染色体的方法。这是因为单倍型对于鉴定致病基因座更有效。为了实现这一目标，我们确定了三个具体目标。 目标1.我们将开发一种双末端rolony测序策略。Rolonies是滚环扩增子菌落。Rolonies是乳液PCR珠的非常有前途的替代品。Rolonies是有吸引力的，由于其简单和小尺寸。然而，配对末端rolony测序目前不可能使用合成测序，并且我们强烈认为配对末端读取对于允许人类基因组的完整单倍型解析组装是必不可少的。因此，目标1致力于建立双端测序方法。我们的配对末端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，我们将在我们的ISFET电测序装置中使用不含荧光标记的可逆终止子应用并优化配对末端测序策略。 目标2.我们将开发一种测序策略，允许组装人类基因组，同时保持单倍型信息。我们将我们的方法称为单倍型分辨全基因组测序（hrWGS）。我们相信，人类基因组序列信息是最有价值的，单体型必须确定。单倍型对于鉴定遗传变异和疾病之间的关联是重要的。此外，单倍型信息是群体遗传分析的关键因素。目前，没有一种下一代测序技术被设计用于提供单倍型信息。我们提出了两种单倍型分辨测序的策略。 目标3。我们将开发一种基于非光的双端克隆测序策略。基于非光的策略将采用高密度ISFET阵列来检测当在合成测序期间掺入碱基时发生的生物化学事件。我们的非光测序策略比现有技术有了巨大的改进。也就是说，我们将联合收割机rolony测序与创新的ISFET设计相结合，以允许每个芯片10 B传感器。此外，我们将利用我们创新的双端测序策略和新的文库 制备方法以允许单体型解析。 总之，我们的创新技术提供了超越目前可用方法的重大进步，并将大大推进当前的最先进技术。我们估计，我们可以测序单倍型解决人类基因组远低于1,000美元使用拟议的技术。

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.