The ultimate goal of this project is to develop an extremely compact and inexpensive DNA sequence for applications in laboratory and clinical settings. The device will implement pyrosequencing on a disposable droplet based "digital microfluidic" cartridge and will be capable of sequencing 10's to 100's of nucleotides at a very low cost per run and at a low initial equipment investment. This platform will also integrate sample preparation, including DNA amplification, with sequencing to provide the integration and ease-of-use demanded of clinicians and other non-specialists who require DNA sequence data. The initial application for this capability will be microbial pathogen identification. In Phase I, a prototype was developed and the microfluidic protocols and biochemical reaction conditions for pyrosequencing were optimized. As a feasibility demonstration 20-40 base pairs within the ITS2 genetic region were sequenced for 13 different molds and yeasts which provided unambiguous identification of each isolate. In Phase II, this success will be built upon by integrating sample preparation steps including DNA concentration, DNA amplification, and template preparation into the system. The integrated system will be evaluated and compared to other commercially available solutions in terms of speed, cost and accuracy of fungal identification. Further optimization of the pyrosequencing biochemistry will be performed to enable longer read lengths on shorter time scales while retaining high accuracy. Finally, a data analysis tool will be developed to automate base calling and database queries. The final product will represent a streamlined approach to fungal species identification, integrating a number of components of the contemporary pathogen identification workflow.

Public Health Relevance

A compact and inexpensive instrument which can automatically identify fungal isolates by sequencing a small region of their DNA will be developed and tested. This product would greatly improve the ability of clinicians to treat infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
5R44HG005186-03
Application #
8337332
Study Section
Special Emphasis Panel (ZRG1-IMST-J (15))
Program Officer
Schloss, Jeffery
Project Start
2009-09-30
Project End
2013-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$852,715
Indirect Cost
Name
Advanced Liquid Logic
Department
Type
DUNS #
140695474
City
Research Triangle Park
State
NC
Country
United States
Zip Code
27709