SBIR-STTR Award

Encouraged by NIH's challenge to enable the $1000 genome, current developments in massively parallel sequencing (MPS) continue to increase throughput and reduce the cost of whole-genome sequencing. Unfortunately, the corresponding sample preparation methodologies now lag behind in throughput and DNA quality, thus slowing the pace of discovery and adoption of sequencing techniques in clinical diagnostics. This proposal's focus is to demonstrate the feasibility of controlled DNA shearing utilizing miniature (<200¿L sample) very high pressure (up to 400Mpa) single pass discharge under controlled and highly energetic conditions to generate the input sample for future massively parallel sequencing (MPS). The proposed systems build on established hydrodynamic mechanism of fluid shear generated under high differential pressure nozzle flow. Long DNA fragments traversing though the nozzle's high velocity gradient are pulled apart due to intense viscous drag forces. For example, at these high pressures, fluid flow velocity will reach 3 times the speed of sound within a very short transition distance. By varying the control parameters of pressure and flow, back pressure, different levels of fragmentation should be achieved. This fragmentation process is not probabilistic and acts upon every DNA molecule that transits the nozzle. The Phase I proposal is focuses on the optimization of control parameters to achieve desired DNA fragmentation performance, following up on preliminary work already done on several prototypes of the two alternative approaches to nozzle design described herein. Each of these approaches can be multiplexed for high throughput use. During Phase II, we intend to expand our studies into large parallel process demonstration.

Public Health Relevance Statement:


Public Health Relevance:
The proposed study aims to develop a high-throughput high pressure system for automated sample preparation for Next Generation Sequencing and other applications to facilitate better control of DNA shearing while minimizing losses and further reducing costs. Higher yields of DNA fragments of desired length, and less potential chemical DNA damage, are expected to improve DNA sequencing-based personalized diagnostics and lead to considerable benefits in healthcare and biomedical research fields.

Project Terms:
Address; Adoption; Animal Model; Back; base; Biomedical Research; Cells; Chemicals; Clinical; Confined Spaces; cost; Cultured Cells; Cytolysis; design; Development; Devices; Diagnostic; DNA; DNA Damage; DNA Fragmentation; DNA Sequence; Elasticity; Ensure; Epigenetic Process; Equilibrium; experience; fluid flow; Future; Genome; genome sequencing; Genomics; Health Services Research; Human Microbiome; improved; Industry; instrumentation; Lead; Length; Liquid substance; Methodology; Methods; microbiome; miniaturize; next generation sequencing; Organism; parallel processing; Performance; Phase; Polymers; Preparation; pressure; prevent; Process; Property; prototype; public health relevance; Robotics; Role; Sample Size; Sampling; scale up; seal; Silicones; Solutions; sound; Speed (motion); System; Systems Development; Techniques; Technology; Temperature; Testing; Time; Travel; Tube; Viscosity; Work; Yeasts

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

Public Health Relevance Statement:


Public Health Relevance:
The aim of this Phase II proposal is to develop a high-throughput, high pressure, automatable, hydrodynamic DNA shearing device prototype. This device is designed to improve control of DNA fragmentation and minimize losses and potential chemical DNA damage, while reducing costs for Next Generation Sequencing and other applications. More efficient generation of DNA in the desired size range(s) will improve DNA sequencing-based personalized diagnostics and lead to considerable benefits in biomedical research and healthcare.

Project Terms:
active control; Adoption; Automation; Bacteria; base; Biological Preservation; Biomedical Research; Caliber; cell type; Cells; Chemicals; Collection; Computer software; cost; Cytolysis; design; Development; Devices; DNA; DNA Damage; DNA Fragmentation; DNA Repair; DNA Sequence; Drops; ds-DNA; Elements; Ensure; Equilibrium; Equipment; Feedback; Generations; genome sequencing; Genomic DNA; Geometry; Goals; Health; Healthcare; Hydrostatic Pressure; improved; Injection of therapeutic agent; instrument; instrumentation; Laboratories; Lead; Ligation; Liquid substance; Mechanics; Methods; miniaturize; Monitor; next generation sequencing; Performance; Phase; Polishes (substance); Preparation; pressure; Process; programs; Protocols documentation; prototype; Pump; Readiness; Recovery; research and development; Running; Sampling; Secure; Sequence Analysis; Site; software development; Staging; System; Techniques; Technology; Temperature; Testing; Time; Tissues; tool; Validation; Vendor; Viscosity; Yeasts