High Pressure Sample Preparation Instrumentation for DNA Sequencing

描述（由申请人提供）：下一代测序（NGS）的发展继续提高全基因组测序的吞吐量并降低成本。不幸的是，DNA制备方法在通量和DNA质量上都落后，减缓了采用新测序技术的步伐。第二阶段提案的重点是开发一种自动化的商业原型DNA剪切装置，以满足许多流行的和新兴的NGS平台的要求。在第二阶段，我们将开发一种微型自清洁主动剪切喷嘴，并将其与恒压泵集成在一起，以实现流动剪切设计，并易于样品导入和收集。此外，我们将以第一阶段的进展为基础，优化最佳DNA剪切所需的参数范围（压力、流速、孔口直径和设计），以产生可预测的窄尺寸范围内的片段。我们将制造两个原型仪器，放置在我们的研发实验室和合作地点，并将描述自动化系统原型的性能。我们已经确定了关键系统组件的供应商，并确保了学术合作者来验证新平台的性能。我们希望在第二阶段结束时实现生产准备。在Specific Aim #1中，我们将开发一种微型自动自清洁主动剪切喷嘴，其次是Specific Aim #2，我们将把剪切喷嘴与恒压泵以及样品导入和收集手段集成在一起。在这个目标中，我们还将开发计算机软件，以方便主动喷嘴和样品管理的自动控制。在具体目标#3中，我们将确定所提出的集成系统最佳性能所需的参数范围（压力，流量，温度和粘度）。在Specific Aim #4中，我们将描述DNA剪切应用程序的自动化系统原型的性能，并开发相应的应用程序。最后，在Specific Aim #5中，我们将评估该系统对细胞裂解和均质化的适用性，并为此目的优化硬件组件。我们已经确定了大多数关键组件的供应商，并确保了学术合作者来验证新平台的性能，因此我们希望在第二阶段结束时实现制造准备

DESCRIPTION (provided by applicant): Developments in next generation sequencing (NGS) continue to increase throughput and reduce the costs for whole-genome sequencing. Unfortunately, DNA preparation methods lag behind in both throughput and DNA quality, slowing the pace of adoption of new sequencing techniques. This Phase II proposal focuses on the development of an automated, commercial prototype DNA shearing device that matches the requirements of many popular and emerging NGS platforms. In Phase II we will develop a miniature self-cleaning active shearing nozzle and integrate it with a constant pressure pump to allow for a flow-through shearing design and easy sample introduction and collection. In addition we will build on our progress from Phase I to optimize the range of parameters (pressure, flow rate, orifice diameter and design) necessary for optimal DNA shearing to generate fragments in predictable, narrow size ranges. We will manufacture two prototype instruments for placement at our R&D laboratory and at a collaborator site, and will characterize the performance of the automated system prototypes. We have identified vendors for critical system components and secured academic collaborators to validate the performance of the new platform. We expect to achieve manufacturing readiness by the conclusion of this Phase II period. In Specific Aim #1 we will develop a miniature automatic self-cleaning active shearing nozzle, followed by Specific Aim #2 where we will integrate the shearing nozzle with a constant pressure pump and the means of sample introduction and collection. In this Aim we will also develop computer software to facilitate automatic control of the active nozzle and sample management. In Specific Aim #3 we will identify the range of parameters (pressure, flow, temperature, and viscosity) necessary for optimal performance of proposed integrated system. In Specific Aim #4 we will characterize the performance of the automated system prototypes for DNA shearing applications and develop corresponding applications. Finally in Specific Aim #5 we will evaluate the suitability of the system for cell lysis and homogenization and optimize the hardware components for that purpose. We have identified vendors for most critical components and secured academic collaborators to validate performance of the new platform, therefore we expect to achieve the manufacturing readiness by the conclusion of this Phase II period