Microfluidic DNA Sequencing

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。 与公共卫生相关：在这个项目的第二阶段，我们将扩大我们在第一阶段完成的过程，以制造一台能够以不到1000美元的价格对人类基因组进行测序的机器。这是一个项目，旨在开发一种用户友好的桌面仪器，可以在大约6小时内对一个人的整个基因组成进行测序；整个过程包括分析在内的成本将不到1000美元。通过了解一个人的基因构成，研究人员和医生可以利用这些信息做出个性化的决定，从而改变医疗保健格局，改善医疗保健成本和结果。这一仪器将极大地提高全球数千万人的医疗水平，并极大地提高我们了解身体功能的能力，这可能会导致医学上的重大突破。

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.