Haplotype Resolved Sequencing Technology

描述(申请人提供)：项目摘要：这项建议的重点是开发一种电子/非光学下一代测序技术，以低于1,000美元的成本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，使研究人员有能力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，单倍型的重要性在很大程度上被忽视了。为了真正了解一种特定疾病的遗传构成，确实需要开发方法来识别所有多态的特定染色体。这是因为单倍型在识别致病基因方面更有效。为了实现这一目标，我们确定了三个具体目标。目的1.我们将开发一种配对末端RORONY测序策略。罗洛尼是滚动的圆形扩增菌落。RORONIES是一种非常有前途的乳胶聚合酶链球的替代品。劳力士之所以吸引人，是因为它们的简单和小巧。然而，目前还不可能使用合成测序法进行配对末端测序，我们坚信配对末端阅读对于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致力于建立配对末端测序方法。我们的配对末端罗兰测序策略将使用连接到可切割荧光部分的可逆终止子来开发和测试。然后，我们将在我们的ISFET电子测序设备中使用不带荧光标记的可逆终止子来应用和优化成对末端测序策略。目的2.我们将开发一种测序策略，允许在保持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分辨全基因组测序(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和疾病之间的联系很重要。此外，单倍型信息是群体遗传分析中的一个关键因素。目前，没有一种下一代测序技术被设计用于传递单倍型信息。我们建议开发两种单倍型分辨测序策略。目的3.我们将开发一种用于配对末端罗兰的非LIGH测序策略。非基于光的策略将使用高密度ISFET阵列来检测在合成测序过程中结合碱基时发生的生化事件。我们的非光测序策略在现有技术的基础上提供了巨大的改进。也就是说，我们将把罗洛尼测序与创新的ISFET设计相结合，以支持每个芯片10B传感器。此外，我们将利用我们创新的配对末端测序策略和新奇的文库 允许单倍型解析的制备方法。总而言之，我们的创新技术提供了比目前可用的方法更大的进步，并将显著推进当前的最先进水平。我们估计，使用拟议的技术，我们可以用远低于1,000美元的成本对单倍型解析的人类基因组进行测序。

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.