Tunable, narrow molecular weight distribution DNA for nanopore sequencing

用于纳米孔测序的可调窄分子量分布 DNA

• 批准号: 10412055
• 负责人: Kevin D Dorfman
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412055
• 关键词: Address; Automobile Driving; Bacteriophage T4; Biological Models; Cells; Centrifugation; Complement; Complex; Custom; DNA; DNA Damage; DNA Sequence; DNA sequencing; Data; Data Set; Devices; Disease; Disease Outbreaks; Equilibrium; GTP-Binding Protein alpha Subunits, Gs; Gel; Genome; Genomics; Glean; Goals; Gold; Human; Laboratories; Lead; Length; Location; Mission; Modeling; Molecular; Molecular Target; Molecular Weight; Motor; National Human Genome Research Institute; Needles; Physics; Polymers; Preparation; Probability; Protocols documentation; Public Health; Pump; Reporting; Reproducibility; Research; Resources; Rheology; Running; Sampling; Syringes; System; Tail; Techniques; Technology; Testing; Time; Translating; Tube; Variant; Weight; Width; base; design; ds-DNA; flexibility; gel electrophoresis; improved; innovation; insight; meetings; nanopore; new technology; next generation sequencing; novel strategies; personalized medicine; prototype; remote location; screening; simulation; tool

Summary Nanopore sequencing is at the cutting-edge of the DNA sequencing revolution, providing long reads that greatly facilitate genome assembly and identify structural variations. The platform’s flexibility and low barrier to entry make it attractive for remote locations, for rapid analysis during disease outbreaks, and for routine sequencing in laboratories that do not have easy access to, or the need for, large-scale centralized sequencing resources. However, nanopore sequencing is biased towards short DNA owing to transport limitations in the device. The polydispersity of the initial molecular weight distribution of the double-stranded DNA (dsDNA) becomes crucial and typically determines the read lengths. Achieving long read lengths requires controlled breakage of megabase genomic dsDNA into smaller fragments with narrow size distributions with a high average molecular weight. The preferred approach is flow-based scission, which yields sequence-independent break points with low DNA damage. The state-of-the-art, developed 20 years ago, pumps the dsDNA many times through a contraction, with commercial devices producing 90% of the molecules within a factor of 2x of the target weight. Other commonly used approaches include multiple passes through a syringe needle, which results in poorly controlled in molecular weight distributions, or centrifugation in a g-tube, which only accesses lower molecular weights. There is considerable room for improvement on the state-of-the-art for dsDNA scission for nanopore sequencing sample preparation, both in terms of the target molecular weights and, more importantly, the distribution about that target weight. This exploratory R21 project will address the unmet need in nanopore sequencing for DNA samples with a narrow distribution about a tunable target molecular weight. Meeting this need would allow users to balance their relative desire for throughput versus read length, while achieving read-length reproducibility between sequencing runs. The goal is to produce a prototype device and protocols that target molecular weights of 30 kilobases (for standard nanopore sequencing), 70 kilobases (for long-read sequencing), and 100 kilobases (for ultra-long read sequencing), with at least 95% of the molecules within 1.5x of the target weight and < 10% variation between runs. The successful completion of this project will establish the feasibility of using flow to provide a relatively simple, inexpensive device with unprecedented tunability of the target DNA molecular weight and an exceptionally narrow size distribution compared to the state-of-the-art. This project is significant because it will provide a new tool in the nanopore sequencing pipeline, complementing ongoing improvements in the sequencing technique itself by addressing a critical need in sample preparation. The project is innovative in its leveraging of concepts in polymer physics and non-Newtonian rheology to improve genomics.

总结 纳米孔测序处于DNA测序革命的前沿，提供长读段， 促进基因组组装和识别结构变异。平台的灵活性和低进入门槛 使其对偏远地区、疾病爆发期间的快速分析和常规测序具有吸引力 在不容易获得或不需要大规模集中测序资源的实验室中。 然而，由于装置中的运输限制，纳米孔测序偏向于短DNA。的 双链DNA（dsDNA）的初始分子量分布的多分散性变得至关重要 并且通常确定读取长度。实现长读取长度需要受控的兆字节断裂 将基因组dsDNA切割成具有窄尺寸分布和高平均分子量的较小片段。的 优选的方法是基于流动的断裂，其产生具有低DNA的序列独立断裂点 损害20年前开发的最先进技术，通过收缩多次泵入dsDNA， 商业装置产生90%的分子，其在目标重量的2倍内。其他 通常使用的方法包括多次穿过注射器针头，这导致控制不良。 在分子量分布中，或在G管中离心，其仅获得较低的分子量。 在用于纳米孔测序的dsDNA切割的最新技术水平上存在相当大的改进空间 样品制备，无论是目标分子量，还是更重要的是，分布 这个目标体重。 这个探索性的R21项目将解决DNA样品纳米孔测序中未满足的需求， 关于可调目标分子量的分布。满足这一需求将允许用户平衡其相对 期望通量与读取长度，同时实现测序运行之间的读取长度再现性。 目标是产生靶向30种酶的分子量的原型装置和方案（用于 标准纳米孔测序）、70个内切酶（用于长读取测序）和100个内切酶（用于超长读取 测序），其中至少95%的分子在目标重量的1.5x内，并且在目标重量与目标重量之间的变化< 10%。 跑步。该项目的成功完成将确立使用流量的可行性，提供一个相对 一种简单、廉价的装置，具有前所未有的靶DNA分子量的可调性， 与最先进的技术相比，尺寸分布非常窄。该项目意义重大，因为它将 在纳米孔测序管道中提供新的工具，补充了正在进行的改进， 测序技术本身解决了样品制备中的关键需求。该项目是创新的， 利用聚合物物理学和非牛顿流变学的概念来改进基因组学。