DNA barcoding via multi-scan and step control in dual-pore tug-of-war

通过双孔拔河中的多重扫描和步骤控制进行 DNA 条形码

• 批准号: 10027758
• 负责人: William Bruce Dunbar
• 金额: $ 47.66万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10027758
• 关键词: Address; Algorithms; Back; Bar Codes; Benchmarking; Binding; Binding Proteins; Calibration; Complex; DNA; Detection; Devices; Disease; Ensure; Equipment; Face; Funding; Genome; Genomic DNA; Genomic approach; Genomics; Individual; Knowledge; Length; Link; Logic; Maps; Measures; Molecular; Molecular Conformation; Output; Pattern; Phase; Physics; Positioning Attribute; Prevalence; Process; Reagent; Regulatory Element; Sampling; Scanning; Speed; System; Technology; Testing; Time; War; Work; base; cost; design; design and construction; ds-DNA; experimental study; functional genomics; genetic regulatory protein; genome-wide; nanopore; programs; response; scaffold; sensor; simulation; single molecule; solid state; tool

Background and Significance: Precise mapping of the binding position of molecular motifs along long, individual dsDNA strands in highly heterogeneous samples is core to a wide range of genomics applications “beyond” sequencing. One candidate approach for molecular feature mapping is based on measuring modulations in the ionic current arising when a dsDNA is electrically driven through a solid-state nanopore (ss-nanopore). Nanopores are attractive as they have a purely electrical read- out, leading to a small foot-print and substantial cost reductions. Recent work demonstrates that ss- nanopores have sufficient sensitivity to detect a wide-range of molecular motifs on translocating dsDNA. Yet, fundamental challenges continue to hinder genome scaling of solid-state nanopore technology: (1) the need to ensure consistent linearization of translocating molecules, (2) need to reduce effect of molecular fluctuations that introduce random error and (3) need to develop strategies to perform accurate genomic distance calibration. Exploiting our recent work on DNA control using devices with two closely separated ss-nanopores, we will address challenges (1)-(3) and obtain feature barcodes from dsDNA possessing sufficient quality to permit genome-scale alignment of individual molecule reads. This is a critical step to enable application of ss-nanopore sensing to heterogenous genomic DNA samples where every molecule sensed in the device can have a different underlying sequence. Technical Approach: A DNA molecule will be threaded simultaneously through two closely separated pores and caught in a molecular “tug-of-war.” Tug-of- war leads to rapid DNA linearization (addressing challenge 1); in addition, independent distance calibration can be performed by measuring the time-of-flight (TOF) of a molecular feature between the pores (addressing challenge 3). Using active logic based on a Field-Programmable Gate Array (FPGA), we can change the molecule’s translocation direction in response to detecting passage of molecular features. This enables back-and-forth rescanning of a local DNA region that can be used to increase precision through averaging (addressing challenge 2). Specific Aims: we will first benchmark accuracy of genomic distance prediction using designed constructs with two features of known spacing (AIM1); we will then extend our two-feature mapping strategy to multi-feature profiles via multi-scan and step control (AIM2); and finally apply multi-feature profiling to heterogeneous samples containing DNA fragments from Mbp-scale genomes (AIM3). The final deliverable is a ss- nanopore based platform that can align feature barcodes to Mbp scale genomes. Further funding phases will drive scaling to Gbp-size genomes and develop multiplexed mapping strategies that combine a barcoding motif with additional molecular motifs (e.g. regulatory proteins to provide a functional annotation/overlay relative to the sequence scaffold established by the barcoding motif).

背景和意义：沿着分子基序的结合位置的精确映射 高度异质样品中的长长的单个dsDNA链是广泛的基因组学的核心 应用“超越”测序。一种分子特征映射的候选方法是基于 在测量DSDNA通过A电气驱动时产生的离子电流中的调节 固态纳米孔（SS纳米孔）。纳米孔具有吸引力，因为它们具有纯电读取 - 出去，导致脚印小，成本降低。最近的工作表明SS- 纳米孔具有足够的灵敏度，可以检测到易位的大量分子基序 dsdna。然而，基本挑战继续阻碍固态纳米孔的基因组规模 技术：（1）需要确保易位分子的线性化一致，（2）需要 减少引入随机误差的分子波动的影响，（3）需要制定策略 进行准确的基因组距离校准。利用我们最近使用DNA控制的工作 具有两个紧密分开的SS纳米孔的设备，我们将解决挑战（1） - （3）并获得 DSDNA的特征条形码具有足够的质量，可以允许基因组规模对齐 单个分子读取。这是启用SS-Nanopore感应的关键步骤 异源基因组DNA样品，其中每个分子中的每个分子都可以具有 不同的基础序列。技术方法：DNA分子将被螺纹 类似地，通过两个紧密分离的孔，并以分子“拖船”捕获。 战争导致快速DNA线性化（应对挑战1）；另外，独立距离 可以通过测量分子特征的飞行时间（TOF）进行校准 毛孔（解决挑战3）。使用基于现场编程的门数组的活动逻辑 （fpga），我们可以改变分子的易位方向，以响应检测通过 分子特征。这使得可以用于来源的当地DNA区域的来回撤销 通过平均（解决挑战2）提高精度。具体目的：我们将首先 基因组距离预测的基准准确性，使用设计的构造具有两个特征 已知间距（AIM1）；然后，我们将把我们的两种映射策略扩展到多功能配置文件 通过多扫描和步骤控制（AIM2）；最后将多功能分析应用于异质 包含来自MBP尺度基因组的DNA片段的样品（AIM3）。最后的交付是SS- 基于纳米孔的平台，可以将特征条形码与MBP刻度基因组保持一致。进一步的资金 阶段将推动缩放为GBP大小的基因组，并制定多路复用的映射策略 将条形码基序与其他分子基序相结合（例如，调节蛋白提供A 功能注释/覆盖相对于条形码基序建立的序列支架）。