High Accuracy Nanopore Sequencing.

项目摘要 该提案的目的是释放纳米孔测序的全部潜力。 这种测序方法具有变革性的内在品质，例如长读取长度， 直接的表观遗传修饰检测，快速的样品到答案，便携性和低成本- 但相对较低的测序准确度仍然是一个显著的缺点。 在NHGRI的资助下，我们建立了一个团队， 开发纳米孔测序，首先工程化高度敏感的孔MspA， 提供单核苷酸分辨率，然后将其与酶控制相结合， 纳米孔测序的首次概念验证。最近，我们开发了一种混合动力- 电压-酶控制方法，其提供了纳米孔的显著增加 测序准确性。同时，我们开发了一种纳米孔单分子工具， 测量酶的动力学，这些酶以前所未有的速度沿着沿着DNA或RNA移动， 详细 在这里，我们建立在我们以前的研究，并提出了有根据的和创新的 进一步提高纳米孔测序准确性的方法， 单次碱基识别准确率约为99%。 我们的具体目标是（1）使用序列依赖性酶动力学来创建一个 纳米孔系统中的第二读取器，其与离子电流读取器串联运行。 该阅读器将提供与离子电流无关的序列信息 读者我们将设计具有增强序列依赖动力学的酶。（二） 我们将系统地绘制序列依赖性酶动力学， 信息转化为新的碱基调用算法。此外，我们将测量离子 孔-酶-DNA复合物的所有四个不同方向的电流，以最大化 这种新方法的准确性。(3)我们将设计出更好的序列特异性， 通过创建允许不对称组装的坚固平台 的MspA。然后，我们将设计具有不对称孔收缩的MspA，以减少 对DNA的布朗运动和增强核苷酸的识别. 我们的团队迄今为止的成功使我们能够与杰出的 合作者，并获得学术界和工业界许多优秀实验室的支持， 他们的专业知识帮助我们向前迈出每一步。我们将与合作实验室合作， 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.