High Accuracy Nanopore Sequencing.

项目总结 这项提议的目标是充分释放纳米孔测序的潜力。 这种测序方法具有变革性的固有特性，例如较长的读出长度， 直接表观遗传修饰检测、快速样本到答案、便携性和低成本- TO-Entry，但相对较低的测序精度仍然是一个重大缺点。 在NHGRI的资助下，我们建立了一支在 开发纳米孔测序，首先设计高度敏感的孔MSPA以 提供单核苷酸解析，然后将其与酶控制相结合 纳米孔测序的第一个概念验证。最近，我们开发了一种混合动力车- 提供纳米孔大幅增加的电压-酶控制法 测序准确性。同时，我们开发了一种纳米孔单分子工具 测量沿DNA或RNA以前所未有的速度移动的酶的动力学 细节。 在此，我们在前人研究的基础上，提出了有理有据的创新建议 进一步提高纳米孔测序准确度的方法 单通道基本呼叫准确率约为99%。 我们的具体目标是(1)使用序列依赖的酶动力学来创建一种 纳米孔系统中与离子电流读取器串联运行的第二个读取器。 该读取器将提供独立于离子电流的序列信息 读者。我们将设计具有强化的序列依赖动力学的酶。(2) 我们将系统地绘制序列依赖酶的动力学图并将其拼接起来 信息转化为新的基本调用算法。此外，我们还将测量离子 电流的所有四个不同方向的孔-酶-DNA复合体最大限度地 这种新方法的精确度。(3)我们将设计更好的序列特异性 离子电流读取器通过创建允许不对称组装的坚固平台 MSPA的。然后我们将设计具有不对称气孔收缩的MSPA以减少 DNA的布朗运动和增强核苷酸识别。 我们团队到目前为止的成功使我们能够与著名的 合作者，并获得学术界和工业界许多优秀实验室的支持， 他们的专业知识将帮助我们向前迈进每一步。我们将与我们的合作实验室合作 完成本提案中概述的目标。

PROJECT SUMMARY The objective of this proposal is to unlock the full potential of nanopore sequencing. This sequencing method has transformative intrinsic qualities, such as long read length, direct epigenetic modification detection, fast sample-to-answer, portability and low cost- to-entry, but relatively low sequencing accuracy remains a significant drawback. With NHGRI funding, we have built a team that has played a pivotal role in developing nanopore sequencing, first engineering the highly sensitive pore MspA to provide single-nucleotide resolution and then combining it with enzyme control for the first proof-of-concept of nanopore sequencing. Recently, we developed a hybrid- voltage-enzyme control method that provided a substantial increase in nanopore sequencing accuracy. Concurrently, we developed a nanopore single-molecule tool that measures the kinetics of enzymes which move along DNA or RNA at unprecedented detail. Here we build on our previous research and propose well-founded and innovative methods to further increase the accuracy of nanopore sequencing with the ultimate goal of a single-passage base calling accuracy of around 99%. Our specific aims are (1) to use sequence-dependent enzyme kinetics to create a second reader in the nanopore system that runs in tandem to the ion current reader. This reader will provide sequence information that is independent of the ion current reader. We will engineer enzymes with accentuated sequence-dependent kinetics. (2) We will systematically map the sequence-dependent enzyme kinetics and splice this information into novel base calling algorithms. In addition, we will measure the ion current of all four different orientations of the pore-enzyme-DNA complex to maximize the accuracy with this new method. (3) We will engineer better sequence specificity into the ion current reader by creating a robust platform that permits asymmetric assembly of MspA. We will then design MspA with an asymmetric pore constriction to reduce Brownian motion of the DNA and enhance nucleotide recognition. 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 each step forward. We will work with our partner labs to complete the aims outlined in this proposal.