Microfluidic DNA Sequencing

微流控 DNA 测序

• 批准号: 8365730
• 负责人: TAL RAZ
• 金额: $ 149.93万
• 依托单位: GNUBIO, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-14 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365730
• 关键词: Algorithms; Bar Codes; Biological Assay; Color; DNA; DNA Library; DNA Resequencing; DNA Sequence; Data Analyses; Detection; Devices; Drops; Dyes; Funding; Genetic; Genome; Genomics; Health Care Costs; Healthcare; Hour; Human Genome; Informatics; Lead; Length; Libraries; Marketing; Medical; Medicine; Methodology; Methods; Microfluidic Microchips; Microfluidics; Outcome; Persons; Phase; Physicians; Preparation; Process; Protocols documentation; Reading; Reagent; Research Personnel; Running; Schedule; Site; Small Business Innovation Research Grant; Sorting - Cell Movement; System; Systems Analysis; Testing; Time; Variant; Work; base; cost; genome sequencing; improved; instrument; meetings; sensor; synthetic construct; user-friendly

DESCRIPTION (provided by applicant): This is a Phase II SBIR proposal to increase the throughput of our DNA sequencing instrument to enable whole genome sequencing with high efficiency and accuracy for a cost of under $1000 per genome. In Phase I, we developed a working sequencing assay, a microfluidic platform and camera system, and analysis methodology that enable genome alignment and variant calling. We have demonstrated sequencing using both synthetic DNA and genomic DNA amplicons with read lengths up over 600bp and accuracy higher than 99.9% per base. Using internal funds, we are upgrading the Phase I device to become a field ready beta test device for placement into several sites. This will be a single channel device that is capable of inline selection of 250 sequencing targets (average of 200BP long), sequence at 40X coverage, and variant calling in less than 2.5 hours of total run time. The Specific Aim of Phase II is to increase the throughput of this beta test single channel instrument to enable whole genome sequencing with high efficiency and accuracy for a cost of under $1000 per genome. To accomplish this Specific Aim, we will carry out seven Tasks: Task 1: Develop a full-genome sequencing assay protocol for the preparation of a genomic DNA library for sequencing. Task 2: Develop a sensor configuration that can scale to 100s of channels. Task 3: Develop a microfluidic device that can scale to 100s of channels Task 4: Develop a method to read droplet sizes of ~10um Task 5: Develop a sorting assay that sorts amplified DNA from empty drops Task 6: Extend current informatics pipeline for resequencing a whole genome Task 7: Extend current barcode clustering algorithms. At the conclusion of Phase II, we will have a breadboard sequencing system that will be able to demonstrate that sequencing a whole genome in about six hours, including data analysis, genome alignment and variant calling, is fully achievable. The system will support reagent volumes that will meet the per run costs target <$1000. Following Phase II, we expect to use internal funds to have a market ready product within one year. PUBLIC HEALTH RELEVANCE: In Phase II of this project, we will scale the process we have completed in our Phase I to build a machine that will be able to sequence a human genome for less than $1000. This is a project to develop a user-friendly, desktop instrument that can sequence a person's entire genetic make-up in approximately 6 hours; the entire process will cost less than $1000, including analysis. By understanding a person's genetic make-up, researchers and physicians can use the information to make personalized decisions, thereby changing the healthcare landscape and improve healthcare costs and outcomes. This instrument will greatly enhance medical treatments for tens of millions of people around the world and greatly improve our ability to understand how the body functions, which could lead to major breakthroughs in medicine.

描述（由申请人提供）：这是一项II期SBIR提案，旨在提高我们的DNA测序仪器的吞吐量，使全基因组测序具有高效率和准确性，每个基因组的成本低于1000美元。在第一阶段，我们开发了一种有效的测序方法、微流控平台和摄像系统，以及能够实现基因组比对和变异调用的分析方法。我们已经证明了使用合成DNA和基因组DNA扩增子进行测序，读取长度超过600bp，每个碱基的准确度高于99.9%。利用内部资金，我们正在升级第一阶段的设备，使其成为一个现场就绪的beta测试设备，可以放置在几个地点。这将是一个单通道设备，能够在不到2.5小时的总运行时间内内联选择250个测序目标（平均长度为200BP），序列覆盖率为40倍，并调用变体。第二阶段的具体目标是提高该beta测试单通道仪器的吞吐量，使全基因组测序具有高效率和准确性，每个基因组的成本低于1000美元。为了实现这一特定目标，我们将开展七个任务：任务1：制定全基因组测序分析方案，以制备用于测序的基因组DNA文库。任务2：开发可扩展到100个通道的传感器配置。任务3：开发一种可以扩展到100个通道的微流体装置任务4：开发一种读取~10um液滴大小的方法任务5：开发一种从空液滴中分类扩增DNA的分选试验任务6：扩展当前的信息学管道，用于全基因组重测序任务7：扩展当前的条形码聚类算法。在第二阶段结束时，我们将拥有一个面包板测序系统，该系统将能够证明在大约6小时内对整个基因组进行测序，包括数据分析，基因组测序和变体调用，是完全可以实现的。该系统支持的试剂量将满足每次运行成本目标< 1000美元。在二期之后，我们希望在一年内利用内部资金开发出适合市场的产品。