High Accuracy Nanopore Sequencing.

高精度纳米孔测序。

• 批准号: 10631962
• 负责人: JENS GUNDLACH
• 金额: $ 80万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-23 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10631962
• 关键词: Academia; Algorithms; Base Sequence; Complex; Creativeness; DNA; DNA sequencing; Dependence; Detection; Development; Engineering; Enzyme Kinetics; Enzymes; Epigenetic Process; Funding; Gene Duplication; Genomic DNA; Goals; Healthcare; Hybrids; Industrialization; Industry; Ions; Kinetics; Laboratories; Length; Maps; Measures; Methods; Modality; Modification; Molecular Motors; Motion; Motivation; Motor; Movement; Mutation; Mycobacterium smegmatis; National Human Genome Research Institute; Nucleic Acids; Nucleotides; Play; Point Mutation; Polymerase; Principal Investigator; Probability; Program Description; Proteins; RNA; RNA Splicing; Reader; Research; Research Personnel; Resolution; Role; Running; Sampling; Side; Signal Transduction; Specificity; System; Techniques; Technology; Testing; Time; VDAC1 gene; Vertebral column; Walking; Work; base; clinical application; constriction; cost; design; flexibility; helicase; improved; innovation; markov model; motor behavior; mutant; nanoengineering; nanopore; neural network; novel; nucleobase; physical separation; portability; prevent; programs; sequencing platform; single molecule; success; tool; voltage

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.

项目概要 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，例如长读长、 直接表观遗传修饰检测、快速样品到答案、便携性和低成本- 进入，但相对较低的测序准确度仍然是一个显着的缺点。 在 NHGRI 的资助下，我们建立了一支在以下方面发挥了关键作用的团队： 开发纳米孔测序，首先设计高度敏感的孔 MspA 提供单核苷酸分辨率，然后将其与酶控制相结合 纳米孔测序的第一个概念验证。最近，我们开发了一种混合动力 电压酶控制方法使纳米孔大幅增加 测序准确性。同时，我们开发了一种纳米孔单分子工具， 测量以前所未有的速度沿 DNA 或 RNA 移动的酶的动力学 细节。 在此，我们以之前的研究为基础，提出了有理有据且创新的建议 进一步提高纳米孔测序准确性的方法，最终目标 单通道碱基检出准确度约为 99%。 我们的具体目标是（1）利用序列依赖性酶动力学来创建 纳米孔系统中的第二个读取器与离子电流读取器串联运行。 该阅读器将提供独立于离子电流的序列信息 读者。我们将设计具有增强的序列依赖性动力学的酶。 (2) 我们将系统地绘制序列依赖性酶动力学图谱并将其剪接 信息转化为新颖的碱基识别算法。此外，我们还将测量离子 孔-酶-DNA复合物的所有四种不同方向的电流，以最大化 这种新方法的准确性。 (3) 我们将设计更好的序列特异性 通过创建允许不对称组装的强大平台来实现离子电流读取器 MspA。然后，我们将设计具有不对称孔收缩的 MspA，以减少 DNA 的布朗运动并增强核苷酸识别。 我们团队迄今为止的成功使我们能够与知名企业建立合作伙伴关系 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前进的每一步。我们将与我们的合作伙伴实验室合作 完成本提案中概述的目标。

项目成果

Mutual information between discrete and continuous data sets.

• DOI: 10.1371/journal.pone.0087357
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Ross BC

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

• DOI: 10.1093/nar/gkac837
• 发表时间: 2022-10-14
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Craig, Jonathan M.;Mills, Maria;Kim, Hwanhee C.;Huang, Jesse R.;Abell, Sarah J.;Mount, Jonathan W.;Gundlach, Jens H.;Neuman, Keir C.;Laszlo, Andrew H.
• 通讯作者: Laszlo, Andrew H.

DNA sequence-dependent ionic currents in ultra-small solid-state nanopores.

• DOI: 10.1039/c6nr01061j
• 发表时间: 2016-05-05
• 期刊: Nanoscale
• 影响因子: 6.7
• 作者: Comer J;Aksimentiev A
• 通讯作者: Aksimentiev A

Stretching and controlled motion of single-stranded DNA in locally heated solid-state nanopores.

• DOI: 10.1021/nn403575n
• 发表时间: 2013-08-27
• 期刊: ACS NANO
• 影响因子: 17.1
• 作者: Belkin, Maxim;Maffeo, Christopher;Wells, David B.;Aksimentiev, Aleksei
• 通讯作者: Aksimentiev, Aleksei

Subangstrom single-molecule measurements of motor proteins using a nanopore.

• DOI: 10.1038/nbt.3357
• 发表时间: 2015-10
• 期刊: Nature biotechnology
• 影响因子: 46.9
• 作者: Derrington IM;Craig JM;Stava E;Laszlo AH;Ross BC;Brinkerhoff H;Nova IC;Doering K;Tickman BI;Ronaghi M;Mandell JG;Gunderson KL;Gundlach JH
• 通讯作者: Gundlach JH