High Accuracy Nanopore Sequencing

主要研究者/项目负责人（最后，第一，中间）：Gundlach，Jens，H。 高精度纳米孔测序 项目摘要 该提案的目的是使纳米孔测序技术能够充分发挥其 潜力在NHGRI的资助下，我们的团队在开发纳米孔方面发挥了关键作用。 测序我们通过首先设计高灵敏度的孔， MspA提供单核苷酸分辨率，然后通过MspA控制DNA速度 用一种酶来首次证明纳米孔测序的概念。在本申请中，我们 概述技术，将克服最后一个障碍，纳米孔测序，低碱基调用 精度 我们通过此申请请求资助，以提高单通道碱基识别准确性 纳米孔测序超过一个数量级，优于99%。这个目标是一个 这是纳米孔测序的变革性一步，因为它开辟了 这项技术在医疗保健和其他领域的应用。此外，我们还将探索其他改进措施， 使纳米孔测序更便宜和更有用的变化。 我们的具体目标是：（1）实现DNA的混合电压酶控制，使DNA抖动 来回，从而允许识别和校正酶错误模式，而 最大化可从离子电流读数获得的信息，（2）改善基本的 通过（i）改进用于测序的孔和（ii） 与解旋酶专家合作开发DNA控制酶， 优化纳米孔测序质量，（3）开发允许高整合密度的技术 以增加测序通量并使DNA输入量最小化。 我们团队迄今为止的成功使我们能够与杰出的合作者建立伙伴关系， 获得学术界和工业界许多优秀实验室的支持，他们的专业知识有助于我们 将纳米孔测序引入医疗保健。我们将与合作伙伴实验室合作， 本提案中提出的目标。 我们的目标是通过提供革命性的测序技术来改善人类生活质量 准确、快速、便宜的方法。 项目摘要

Principal Investigator/Program director (Last, first, middle): Gundlach, Jens, H. High Accuracy Nanopore Sequencing PROJECT SUMMARY The objective of this proposal is to enable nanopore sequencing technology to reach its fullest potential. With NHGRI funding, our group has played a pivotal role in developing nanopore sequencing. We paved the way to nanopore sequencing by first engineering the highly sensitive pore MspA to provide single-nucleotide resolution and then by controlling the DNA speed through MspA with an enzyme to give the first proof of concept of nanopore sequencing. In this application we outline techniques that will overcome the last hurdle to nanopore sequencing, low base calling accuracy. We request funding through this application to increase the single-passage base calling accuracy of nanopore sequencing by more than an order of magnitude to better than 99%. This goal is a transformative step forward for nanopore sequencing since it opens up ubiquitous applications of this technology in health care and beyond. Additionally, we will explore other improvements and variations that make nanopore sequencing less expensive and more useful. Our specific aims are: (1) implementing a hybrid-voltage-enzyme control of DNA that dithers DNA back and forth, thereby allowing identification and correction of enzyme error modes, while maximizing the information obtainable from the ion current readings, (2) improving the fundamental components of the nanopore sequencer by (i) improving the pore used for sequencing and (ii) teaming up with helicase experts to develop DNA-controlling enzymes specifically engineered to optimize nanopore sequencing quality, (3) developing techniques that permit high integration density of nanopores to increase sequencing throughput and to minimize DNA input quantity. Our team's success to date has enabled us to form partnerships with prominent collaborators and to gain support from many excellent labs in academia and industry, whose expertise assists us in bringing nanopore sequencing into health care. We will work with our partner labs to complete the aims outlined in this proposal. It is our goal to improve the quality of human life by delivering revolutionary sequencing technology that is accurate, fast and inexpensive. Project Summary