Tunable, narrow molecular weight distribution DNA for nanopore sequencing

摘要 纳米孔测序处于DNA测序革命的前沿，提供了大量的长时间读数 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、疾病暴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。 然而，由于设备中的运输限制，纳米孔测序偏向于短DNA。这个 双链dna(DsDNA)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。要实现较长的读取长度，需要对兆数据库进行受控破坏 基因组dsDNA分成大小分布窄、平均相对分子质量高的小片段。这个 首选的方法是基于流的断裂，它产生与序列无关的断裂点，DNA含量较低 损坏。20年前开发的最先进的设备，通过收缩多次泵送dsDNA， 商业设备在目标重量的2倍范围内生产90%的分子。其他 常用的方法包括多次穿过注射器针头，这会导致控制不善 在分子量分布中，或在G管中离心，它只获得较低的分子量。 在纳米孔测序中，dsDNA切割的最新技术还有相当大的改进空间 样品制备，既包括目标分子量，更重要的是，关于 那个目标体重。 这个探索性的R21项目将解决在DNA样本纳米孔测序中未得到满足的需求 目标分子量可调的分布。满足这一需求将使用户能够平衡其亲属 希望获得与读取长度相比的吞吐量，同时实现测序运行之间的读取长度重复性。 其目标是生产一种原型设备和协议，目标是分子量为30千碱基(用于 标准纳米孔测序)、70千碱基(用于长读测序)和100千碱基(用于超长读取 测序)，至少95%的分子在目标重量的1.5倍以内，并且在 跑了。该项目的成功完成将为使用Flow提供一个相对 简单、廉价的设备，具有前所未有的目标DNA分子量可调性和 与最先进的尺寸分布相比，尺寸分布异常狭窄。这个项目意义重大，因为它将 在纳米孔测序管道中提供新的工具，补充正在进行的改进 通过解决样品制备中的关键需求，测序技术本身也得到了改善。该项目具有创新性。 利用聚合物物理和非牛顿流变学中的概念来改进基因组学。

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.