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：精确绘制分子基序的结合位置 高度异质性样品中长的、单独的 dsDNA 链是多种基因组学的核心 “超越”测序的应用是基于分子特征映射的一种候选方法。 测量当 dsDNA 通过电驱动时产生的离子电流的调制 固态纳米孔（ss-纳米孔）很有吸引力，因为它们具有纯电学特性。 最近的工作表明，ss- 可以减少占地面积并大幅降低成本。 纳米孔具有足够的灵敏度来检测易位的各种分子基序 然而，基本的挑战仍然阻碍着固态纳米孔的基因组扩展。 技术：（1）需要确保易位分子的一致线性化，（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 利用我们最近在 DNA 控制方面的工作进行精确的基因组距离校准。 具有两个紧密分离的不锈钢纳米孔的装置，我们将解决挑战（1）-（3）并获得 来自 dsDNA 的条形码具有足够的质量，可以进行基因组规模的比对 这是实现单分子纳米孔传感应用的关键步骤。 异质基因组 DNA 样本，其中设备中感测到的每个分子都可以具有 不同的底层序列：DNA 分子将被串联。 同时通过两个紧密分开的毛孔并陷入分子“拔河比赛”。 战争导致DNA快速线性化（解决挑战1）；此外，独立距离 可以通过测量分子特征之间的飞行时间 (TOF) 来进行校准 使用基于现场可编程门阵列的有源逻辑（解决挑战 3）。 （FPGA），我们可以改变分子的易位方向以响应检测到的通过 这使得能够对局部 DNA 区域进行来回重新扫描，从而可用于 通过平均提高精度（解决挑战 2）。 使用具有以下两个特征的设计结构进行基因组距离预测的基准准确性 已知间距（AIM1）；然后我们将把我们的两个特征映射策略扩展到多特征配置文件 通过多重扫描和步进控制（AIM2），最后将多特征分析应用于异构 包含来自 Mbp 规模基因组 (AIM3) 的 DNA 片段的样本最终交付的是 ss-。 基于纳米孔的平台，可以将特征条形码与 Mbp 规模的基因组对齐 进一步资助。 各个阶段将推动基因组规模扩大到 Gbp 大小，并开发多重映射策略 将条形码基序与其他分子基序相结合（例如调节蛋白以提供 相对于由条形码基序建立的序列支架的功能注释/覆盖）。