Microfluidic DNA Sequencing

微流控 DNA 测序

• 批准号: 8545203
• 负责人: TAL RAZ
• 金额: $ 147.17万
• 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/8545203
• 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; public health relevance; 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.

描述（由申请人提供）：这是一项第二阶段SBIR提案，旨在增加我们的DNA测序仪器的通量，以实现高效率和准确性的全基因组测序，每个基因组的成本低于1000美元。在第一阶段，我们开发了一种工作测序分析，一种微流体平台和相机系统，以及能够实现基因组比对和变异识别的分析方法。我们已经证明了使用合成DNA和基因组DNA扩增子的测序，其读取长度超过600 bp，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，成为一个现场准备beta测试设备放置到几个网站。这将是一个单通道设备，能够在线选择250个测序靶标（平均200 BP长），以40 X覆盖率测序，并在不到2.5小时的总运行时间内进行变异识别。第二阶段的具体目标是增加这种beta测试单通道仪器的通量，以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，我们将开展七项任务：任务1：开发全基因组测序测定方案，用于制备用于测序的基因组DNA文库。任务2：开发可扩展到数百个通道的传感器配置。 任务三：任务4：开发一种读取~ 10 μ m液滴大小的方法任务5：开发一种从空液滴中分选扩增DNA的分选试验任务6：扩展当前用于全基因组重测序的信息学管道任务7：扩展当前条形码聚类算法。 在第二阶段结束时，我们将拥有一个试验板测序系统，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，基因组比对和变异识别，是完全可以实现的。该系统将支持试剂体积，以满足每次运行成本目标<1000美元。在第二阶段之后，我们预计将使用内部资金在一年内推出市场就绪的产品。