Isolation of long DNA for next-generation genomics applications

分离长 DNA 以用于下一代基因组学应用

• 批准号: 9302912
• 负责人: Kevin D Dorfman
• 金额: $ 17.65万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9302912
• 关键词: Academia; Address; Base Pairing; Cell physiology; Cells; Chromosomes; Collaborations; Complex; Computer software; DNA; DNA purification; Device Designs; Devices; Diffuse; Drops; Electrophoresis; Elements; Entropy; Environment; Generic Drugs; Genome; Genome Mappings; Genomic DNA; Genomics; Geometry; Height; Hela Cells; Hour; Human; Human Cell Line; Human Genome; Industrialization; Length; Malignant Neoplasms; Maps; Methodology; Methods; Microfluidic Microchips; Microfluidics; Minnesota; Minor; Mission; National Human Genome Research Institute; Organism; Plants; Preparation; Proteins; Protocols documentation; Public Health; Pulsed-Field Gel Electrophoresis; RNA; Reagent; Recovery; Research; Sampling; Sepharose; Series; Single Nucleotide Polymorphism; Spectrophotometry; System; Technology; Testing; Time; Universities; Variant; base; cell type; cost; design; experimental study; genome sequencing; high reward; high risk; improved; innovation; nanochannel; nanopore; new technology; next generation; next generation sequencing; novel strategies; pathogen; pressure; programs; prototype; residence; single molecule; voltage; web site

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.

总结 基因组学技术正处于另一场革命之中，这一次的重点是分析长而完整的 基因组DNA分子这些长读技术，包括纳米孔测序，基因组测序， 纳米通道中的映射和基于液滴的条形码，旨在缓解下一阶段的短读长问题， 代测序（NGS）。长读长技术，无论是单独使用还是与NGS结合使用， 代表了一个变革性的突破，解决了基因组测序，组装， 和分析随着长读技术开始成熟，其进一步发展的瓶颈正在移动 样品制备步骤。虽然开发长时间阅读技术需要巨大的创新， 获得长DNA分子的方法，进入这些新的设备已经看到很少 创新提取基因组DNA的标准方法是将细胞包埋在琼脂糖塞中， 提取DNA这项技术是30多年前开发的，用于通过脉冲场凝胶进行染色体大小测定。 电泳，今天仍然是最先进的，只有微小的增量进展。 该提案提供了一种通用方法，可用于为任何长读段创建长DNA样本。 技术.我们项目的创新在于认识到微流体技术可以大大减少反应器 体积的DNA提取，从而大大减少了处理时间，同时保持所需的产量 用于基因组学。在特定目标1中，我们将开发一种基于消除扩散限制的微流体系统 从细胞中提取DNA，将处理时间缩短100倍。在具体目标2中，我们将开发一个 电泳方法回收DNA，相对于现有技术的方法再次减少了时间， 超过一个数量级。在具体目标3中，我们将结合联合收割机的优化设计，从以前的目标 证明从人细胞系中回收DNA，并表明这些DNA具有足够的质量， 纳米通道中的基因组图谱。总之，我们平台的创新功能将减少基因组 DNA提取从劳动密集型，一天的协议到自动化，一小时的协议。这个项目 利用明尼苏达大学与 BioNano Genomics（BNG）成立于过去三年，利用了两者的独特功能 团队微流体装置的设计将在明尼苏达州进行，那里有装置的专业知识。 制造和原型制作。该设备在真实环境中的测试将在BNG进行， 在人类细胞纳米通道基因组图谱方面有专门知识。