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

这项提议旨在实现一种直接低投入单分子DNA和RNA测序的设备。 该装置可用于处理皮卡输入水平，即对应于 单个细胞。读出技术基于单分子实时(SMRT)测序，其中 DNA分子的复制是由聚合酶制造的，从四个碱基进行颜色的光学检测是 用来实时读取序列。除了DNA测序，碱基修饰的检测还包括 可以通过测量复制过程中碱基掺入的动力学来实现。这项技术已经被 由800多种出版物记录，最近，完全基于SMRT的人类基因组组装 报告了测序数据。SMRT测序和基于纳米孔的测序是两种主流 今天的单分子测序技术，既提供长读数长度，也提供直接DNA读数 原生样本。然而，这两种技术都不兼容皮卡级别的输入DNA，这是一个严重的问题 对单细胞或细针活检分析的限制。最后，直接进行RNA测序是非常有意义的 对实现不需要cdna转换和扩增的直接转录组分析以及对探测的兴趣 RNA碱基修饰，但目前还没有可用的方法可以直接对RNA进行测序。 在东北大学的Wanunu小组和太平洋生物科学公司之间的合作中 (PacBio)，我们结合了我们团队在酶工程、有机合成、材料 科学、纳米制造、表面化学和单分子光学检测，以解决上述问题 通过允许从皮克级别的输入直接对DNA和RNA进行测序，这是一个挑战。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 二次结构。通过2012年收到的上一笔R21赠款，Wanunu集团(与 PacBio)已经开发出纳米孔零模波导，并进一步展示了它们在有效捕获 DNA和RNA的皮卡级别，以及对长DNA分子进行测序。在这份提案中，我们描述了一种新的 一种称为多孔零模波导(PZMW)的器件，它将解决以前 器件的设计和可扩展性，以降低制造成本至少2个数量级。在……里面 此外，基于有希望的初步结果，我们将开发一种方法，允许直接皮卡级别 RNA测序。在这种方法中，将使用酶工程来允许RNA模板分子 在PZMW内复制，使全长RNA转录本能够直接测序，并对 二级结构和碱基修饰。我们将演示来自不同低端的DNA和RNA测序 来自我们的合作者的输入来源。拟议研究的成功将提供皮克级的研究 具有长读数长度的DNA和RNA分析，这将通过使更深层次的 了解疾病和细胞发育中的基因组、转录和表观基因组变异。

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.