Isolation of long DNA for next-generation genomics applications

摘要 基因组学技术正处于又一场革命之中，这一次专注于分析长的、完整的 基因组DNA分子。这些久负盛名的技术，包括纳米孔测序、基因组 纳米通道中的映射和基于液滴的条形码旨在缓解Next-Next中的短读取长度问题 世代排序(NGS)。单独使用或与NGS结合使用的长读取长度技术， 代表着一项革命性的突破，解决了目前基因组测序、组装、 和分析。随着长阅读技术开始成熟，它们进一步发展的瓶颈正在移动 至样品制备步骤。虽然开发长篇阅读技术需要巨大的创新， 获得进入这些新设备的长DNA分子的方法还很少见 创新。提取基因组DNA的标准方法是将细胞嵌入琼脂糖塞中，然后 提取DNA。这项技术是在30多年前开发的，用于通过脉冲场凝胶测定染色体大小 电泳法仍然是当今最先进的技术，只有很小的进步。 该建议提供了一种通用方法，可用于为任何长时间阅读创建长DNA样本 技术我们项目的创新之处在于认识到微流体可以大大减少反应堆 DNA提取的体积，从而大大减少处理时间，同时保持所需的产量 为了基因组学。在具体目标1中，我们将开发一种基于微流控系统的、消除扩散限制的系统 在从细胞中提取DNA方面，处理时间减少了100倍。在具体目标2中，我们将制定一项 电泳法回收DNA，与最先进的方法相比，再一次缩短了时间 超过一个数量级。在具体目标3中，我们将结合先前目标的最优设计 展示从人类细胞系中回收DNA，并证明这些DNA具有足够的质量 在纳米通道中绘制基因组图。总而言之，我们平台的创新功能将减少基因组 从一天的劳动密集型程序到一小时的自动化程序提取DNA。这个项目 利用明尼苏达大学和明尼苏达大学之间的创新学术/产业合作 生物纳米基因组学(BNG)，在过去的三年中建立，它利用了两者的独特能力 团队。微流控设备的设计将在明尼苏达州进行，那里有设备方面的专业知识 制造和原型制作。该设备在真实环境中的测试将在BNG进行，在那里 在人类细胞的纳米通道中有基因组图谱方面的专业知识。

Summary Genomics technology is in the midst of yet another revolution, this time focusing on the analysis of long, intact genomic DNA molecules. These long-read technologies, which include nanopore sequencing, genome mapping in nanochannels, and droplet-based barcoding, aim to alleviate the short-read length problem in next- generation sequencing (NGS). Long read-length technologies, either in isolation or combined with NGS, represent a transformative breakthrough that addresses current limitations in genome sequencing, assembly, and analysis. As long-read technologies begin to mature, the bottleneck in their further advancement is moving to sample preparation steps. While tremendous innovations were required to develop long-read technologies, the methodology for obtaining the long DNA molecules that go into to these new devices has seen little innovation. The standard method for extracting genomic DNA is to embed the cells in an agarose plug and extract the DNA. This technology, developed over 30 years ago for chromosome sizing by pulsed-field gel electrophoresis, remains the state-of-the-art today with only minor incremental advances. This proposal provides a generic method that can be used to create a long DNA sample for any long-read technology. The innovation in our project is recognizing that microfluidics can substantially reduce the reactor volume for DNA extraction, and thus massively reduce the processing time, while maintaining the yield needed for genomics. In Specific Aim 1, we will develop a microfluidic system based that removes diffusive limitations in DNA extraction from cells, reducing the processing time by 100-fold. In Specific Aim 2, we will develop an electrophoretic method to recover the DNA, again reducing the time relative to the state-of-the-art method by more than an order of magnitude. In Specific Aim 3, we will combine optimal designs from the previous aims to demonstrate DNA recovery from a human cell line and show that these DNA are of sufficient quality for genome mapping in nanochannels. Taken together, the innovative features of our platform will reduce genomic DNA extraction from a labor intensive, day-long protocol to an automated, hour-long protocol. This project takes advantage of an innovative academia/industrial collaboration between the University of Minnesota and BioNano Genomics (BNG), established over the past three years, that leverages the unique capabilities of both teams. The microfluidic device design will take place at Minnesota, where there is expertise for device fabrication and prototyping. The testing of the device in a real-world environment will take place at BNG, where there is expertise in genome mapping in nanochannels for human cells.