Direct picogram DNA and RNA sequencing using nanopore Zero-mode waveguides

使用纳米孔零模波导直接进行皮克 DNA 和 RNA 测序

• 批准号: 9356545
• 负责人: Meni Wanunu
• 金额: $ 59.8万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-22 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9356545
• 关键词: Access to Information; Address; Architecture; Biological Sciences; Brain; Categories; Cells; Chemistry; Collaborations; Complementary DNA; Complex; Consensus; DNA; DNA analysis; DNA sequencing; DNA-Directed DNA Polymerase; Data; Detection; Development; Device Designs; Devices; Disadvantaged; Disease; Engineering; Enzymes; Epigenetic Process; Funding; Generations; Genes; Genome; Genomic Segment; Genomics; Goals; Grant; High-Throughput DNA Sequencing; Human; Human Genome; Individual; Kinetics; Length; Libraries; Mainstreaming; Measures; Membrane; Methods; Mitochondrial DNA; Modification; Motor; Needle biopsy procedure; Nucleic Acids; Optics; Organic Synthesis; Polymerase; Preparation; Process; Protein Engineering; Publications; RNA; RNA Probes; RNA Processing; RNA Sequence Determination; RNA Sequences; RNA analysis; Reporting; Research; Sampling; Secondary to; Source; Structure; Surface; Technology; Time; Transcript; Universities; Variant; base; color detection; cost; epigenomics; improved; interest; materials science; nanofabrication; nanopore; new technology; programs; scale up; single cell analysis; single molecule; success; transcriptome; transcriptome sequencing; transcriptomics; voltage

This proposal aims at enabling a device for direct low-input single molecule DNA and RNA sequencing. The device can be used to process picogram input levels, amounts that correspond to nucleic acid contents of individual cells. The readout technology is based on single molecule, real-time (SMRT) sequencing, where a copy of a DNA molecule is made by a polymerase enzyme, and optical detection of colors from four bases is employed to read the sequence in real time. In addition to DNA sequencing, detection of base modifications is possible by measuring the kinetics of base incorporation during replication. This technology has been documented by over 800 publications, and recently, a human genome assembly based entirely on SMRT sequencing data was reported. SMRT sequencing and nanopore-based sequencing are the two mainstream single-molecule sequencing technologies today, both offering long read lengths and direct DNA reads from native sample. However, neither technology is compatible with picogram-level input DNA, which is a serious limitation for analysis from single cells or small needle biopsies. Finally, direct RNA sequencing is of great interest for enabling direct transcriptome analysis without cDNA conversion and amplification, and for probing RNA base modifications, but there is no available method that can directly sequence RNA at the present time. In this collaboration between the Wanunu group at Northeastern University and Pacific Biosciences (PacBio), we combine our teams' unique expertise in enzyme engineering, organic synthesis, materials science, nanofabrication, surface chemistry, and single molecule optical detection, to solve the above challenges by allowing direct sequencing of DNA and RNA from picogram levels of input. The methods we will develop also allow for simultaneous detection of epigenetic base modification detection and resolve RNA secondary structures. Through a previous R21 grant received in 2012 the Wanunu group (in collaboration with PacBio) has developed nanopore-zero-mode waveguides, and further showed their use for efficient capture of picogram levels of DNA and RNA, and sequencing long DNA molecules. In this proposal, we describe a new type of device called a porous zero-mode waveguide (PZMW), which will address challenges in the previous device's design and scalability in order to reduce costs of fabrication by at least 2 orders of magnitude. In addition, based on promising preliminary results we will develop a method that will allow direct picogram-level RNA sequencing. In this method, enzyme engineering will be used to allow RNA template molecules to be replicated inside PZMWs, enabling the direct sequencing of full-length RNA transcripts with sensitivity to secondary structure and base modifications. We will demonstrate DNA and RNA sequencing from various low- input sources that come from our collaborators. Success in the proposed research will afford picogram-level DNA and RNA analysis with long read lengths, which would revolutionize genomics by enabling a deeper understanding of genomic, transcriptomic and epigenomic variation in disease and cell development.

该提议旨在实现用于直接低输入单分子DNA和RNA测序的装置。 该装置可用于处理皮克输入水平，即对应于细胞核酸含量的量。 单个细胞。读出技术基于单分子实时（SMRT）测序，其中， 一个DNA分子的拷贝是由聚合酶产生的，从四个碱基的颜色的光学检测是 用来真实的读取序列。除了DNA测序，碱基修饰的检测也是 这可能通过测量复制过程中碱基掺入的动力学来实现。这项技术已经 有超过800篇出版物记录，最近，完全基于SMRT的人类基因组组装 报道了测序数据。SMRT测序和基于纳米孔的测序是两种主流 目前的单分子测序技术，既提供了长的读取长度，又提供了直接的DNA读取， 原生样品然而，这两种技术都不兼容皮克级的输入DNA，这是一个严重的问题。 限制了对单细胞或小针活检的分析。最后，直接的RNA测序是非常重要的。 感兴趣的是能够进行直接转录组分析而无需cDNA转化和扩增，以及用于探测 RNA碱基修饰，但目前还没有可用的方法可以直接测序RNA。 在东北大学的Wanunu小组和太平洋生物科学公司的合作中， （PacBio），我们联合收割机结合我们的团队在酶工程，有机合成，材料 科学、纳米纤维、表面化学和单分子光学检测，以解决上述问题 通过允许从皮克级的输入直接测序DNA和RNA的挑战。我们将采用的方法 开发还允许同时检测表观遗传碱基修饰检测和解析RNA 二级结构。通过2012年收到的R21赠款，Wanunu集团（与 PacBio）开发了纳米孔零模式波导，并进一步展示了它们用于高效捕获 皮克水平的DNA和RNA，以及对长DNA分子进行测序。在本提案中，我们描述了一种新的 一种称为多孔零模波导（PZMW）的设备，它将解决以前的挑战 设备的设计和可扩展性，以便将制造成本降低至少2个数量级。在 此外，基于有希望的初步结果，我们将开发一种方法，将允许直接皮克级 RNA测序。在这种方法中，酶工程将用于允许RNA模板分子被酶解。 在PZMW内复制，使得能够直接测序全长RNA转录物， 二级结构和碱基修饰。我们将展示DNA和RNA测序从各种低- 来自我们合作者的输入来源。这项研究的成功将使皮克级的 具有长读取长度的DNA和RNA分析，这将通过实现更深层次的 了解疾病和细胞发育中的基因组、转录组和表观基因组变异。