High Pressure Sample Preparation Instrumentation for DNA Sequencing

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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