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

背景和意义：精确绘制分子基序沿着的结合位置 在高度异质性的样品中，长的、单个的dsDNA链是广泛的基因组学的核心 应用程序“超越”测序。分子特征映射的一种候选方法是基于 在测量当dsDNA被电驱动通过 固态纳米孔（SS-纳米孔）。纳米孔是有吸引力的，因为它们具有纯粹的电读取- 这导致了一个小的足迹和大幅降低成本。最近的研究表明，SS- 纳米孔具有足够的灵敏度来检测广泛的分子基序， 双链DNA然而，基本挑战继续阻碍固态纳米孔的基因组缩放 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，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， 对于具有两个紧密分离的ss-纳米孔的器件，我们将解决挑战（1）-（3），并获得 来自dsDNA的特征条形码具有足够的质量，以允许基因组规模的比对， 单个分子读数。这是使ss-纳米孔感测能够应用于 异质基因组DNA样本，其中设备中感测的每个分子都可以具有 不同的底层序列技术方法：DNA分子将被 同时通过两个紧密分离的孔，并陷入分子"拔河"。拔河 战争导致快速DNA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， 孔（解决挑战3）。使用基于现场可编程门阵列的有源逻辑 （FPGA），我们可以改变分子的易位方向，以响应检测的通道， 分子特征这使得能够来回重新扫描局部DNA区域，其可用于 通过求平均值提高精度（解决挑战2）。具体目标：我们首先 使用具有两个特征的设计构建体进行基因组距离预测的基准准确度 已知间距（AIM1）;然后，我们将扩展我们的双特征映射策略到多特征轮廓 通过多扫描和步进控制（AIM2），并最终应用多特征分析异构 含有来自Mbp级基因组（AIM 3）的DNA片段的样品。最后的交付物是一个ss- 基于纳米孔的平台，可以将特征条形码与Mbp级基因组对齐。进一步供资 阶段将推动扩展到千兆比特大小的基因组，并开发多路复用的映射策略， 联合收割机将条形码基序与另外的分子基序（例如调节蛋白质）组合，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).