Microfluidic DNA Sequencing

微流控 DNA 测序

• 批准号: 8725992
• 负责人: TAL RAZ
• 金额: $ 149.99万
• 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/8725992
• 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; Genomic DNA; 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SBIR第二阶段的建议，旨在增加我们DNA测序仪的吞吐量，以实现高效率和准确的全基因组测序，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、微流控平台和摄像系统，以及能够实现基因组比对和变体调用的分析方法。我们已经演示了使用合成DNA和基因组DNA扩增进行测序，阅读长度超过600bp，每个碱基的准确率高于99.9%。利用内部资金，我们正在升级第一阶段的设备，以成为现场准备的测试版设备，以便放置在几个地点。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在线选择250个测序目标(平均长度为200个基点)、覆盖40倍的序列以及变体调用。第二阶段的具体目标是增加这种Beta测试单通道仪器的吞吐量，以实现高效率和高精度的全基因组测序，每个基因组的成本低于1000美元。为了实现这一特定目标，我们将执行七项任务：任务1：建立全基因组测序分析方法，用于制备用于测序的基因组DNA文库。任务2：开发可扩展到100秒通道的传感器配置。任务3：开发一种可以扩展到100秒通道的微流控设备任务4：开发一种读取~10um液滴大小的方法任务5：开发一种从空滴中分选扩增DNA的分类方法任务6：扩展现有的信息学流水线，用于对整个基因组进行重新测序任务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，它将能够在大约六个小时内证明整个基因组测序，包括数据分析、基因组比对和变体调用，是完全可以实现的。该系统将支持达到每次运行成本目标1000美元的试剂容量。在第二阶段之后，我们预计将在一年内使用内部基金推出市场就绪的产品。