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

背景和意义：分子基序结合位置的精确定位 高度异质性样本中的长的、单独的dsDNA链是广泛的基因组学的核心 应用程序“超越”排序。分子特征图谱的一种候选方法是基于 关于测量当dsDNA被电驱动时离子电流中的调制 固态纳米孔(ss-Nanore)。纳米孔很有吸引力，因为它们有一个纯粹的电子读数- 从而实现了小规模的占地面积和大幅的成本削减。最近的研究表明，SS- 纳米孔具有足够的灵敏度，可以检测到大范围的分子基序移位 DsDNA。然而，根本性的挑战继续阻碍固态纳米孔的基因组规模。 技术：(1)需要确保运输分子的一致线性化，(2)需要 减少引入随机误差的分子波动的影响，以及(3)需要制定策略 进行精确的基因组距离校准。利用我们最近在DNA控制方面的工作 具有两个紧密分离的ss纳米孔的器件，我们将应对挑战(1)-(3)并获得 来自dsDNA的特征条形码，具有足够的质量，可以进行基因组规模的比对 单个分子读数。这是将固相纳米孔传感应用于 异质基因组DNA样本，在设备中检测到的每个分子都可以有一个 不同的潜在序列。技术方法：DNA分子将被穿线 同时通过两个紧密分离的毛孔，陷入了分子“拔河”。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的飞行时间(TOF)来执行校准 毛孔(应对挑战3)。使用基于现场可编程门阵列的有源逻辑 (现场可编程门阵列)，我们可以改变分子的易位方向，以响应检测到的通过 分子特征。这允许来回重新扫描可用于以下目的的本地DNA区域 通过求平均值来提高精度(应对挑战2)。具体目标：我们将首先 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精度 已知间距(AIM1)；然后我们将把我们的双要素映射策略扩展到多要素剖面 通过多扫描和步长控制(AIM2)；最后将多特征分析应用于异质 含有MBP规模基因组DNA片段的样本(AIM3)。最终的交付成果是一个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。进一步提供资金 各阶段将推动向GBP大小的基因组的扩展，并开发多路复用映射策略 将条码基序与其他分子基序结合(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)。

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).