The 2-4 year objective of our DNA sequencing instrumentation program is to assemble a comprehensive facility that can process and analyze at least 3000 clones per 24 hr per sequencing system. In this application we propose to develop one component of this facility--a highly automated electrophoresis system with integrated base calling software, including appropriate real-time analysis tools. When completed, the electrophoresis system will analyze 384 DNA samples simultaneously with a run-to-run cycle time of less than three hours. It will have a peak sequencing rate of approximately 120,000 bases per hour-and a base read length of at least 500 bases per lane. The electrophoresis system and its associated sample loader will minimize the use of sequencing reagents thereby reducing overall reagent costs. The electrophoresis system we propose will be fully integrated with components being developed under other funded efforts. These other complementary projects include the development of a high-speed sample preparation facility and sequence data management and assembly tools. The specific objectives of this proposal are to develop 1) an automated sample loading system capable of loading 384 samples simultaneously and using 10X less DNA than conventional electro-kinetic injection techniques used for capillary electrophoresis systems; 2) arrays of sealed glass microchannels incorporating 384 separate electrophoresis channels and associated input ports and low-noise optical detection chambers; 3) a fluorescence detection system utilizing a CCD detector for the simultaneous detection of four different spectral bands. 4) the necessary electronics and software for real-time data collection; and 5) integrated base-calling software designed to lengthen base calls and capable of producing rigorous, probability-based assessments of sequence quality. The array of microchannels and sample loader will be built using microfabrication technology to integrate 384 separation columns on glass substrate(s). The laser induced fluorescence detection system will be constructed with an argon-ion laser for excitation and a spectrograph and Charge Coupled Device for four color fluorescence detection. The base calling software will incorporate DNA transport models of peak shape, nobility, and diffusion. Incorporating these models will enable the software to deconvolve runs of bases more reliably, as well as assess the quality of the resulting deconvolutions. The work will be completed in three phases: 1) design optimization; 2) unit construction; and 3) system integration and optimization.
