The goal of this proposal is to develop methods and instrumentation to completely automate the hybridization and detection steps in multiplex sequencing. This is an essential step to increase the throughput and cost-efficiency of multiplex sequencing by a factor of 10 to 20 over the next five years. Research will focus on three areas: Automated high throughput hybridization. Devices which automate the hybridization cycle in multiplex sequencing will be built. The initial machine will be capable of producing more than 50 million bases of raw data per year. Design and testing of the machine will be done within this project so that implementation of the technology into the production project can be done with minimal disruption. Additional machines will be built to allow for longer read length and higher throughput. In year five, a total of six machines will enable raw data generation at a rate of more than 500 million bases per year. Infrared fluorescence detection of multiplex sequences. Methods and instrumentation to use infrared fluorescence labeling and detection for multiplex sequencing will be developed. Protocols will be developed and optimized to label and use hybridization probes carrying single and multiple infrared dye molecules with minimal non-specific binding. Simultaneously, a novel infrared scanning system will be built. Detector components and optical filters will be optimized to reduce noise from membrane scatter and background fluorescence. In addition, phase- and time-resolved detection methods for improved discrimination between signal and background fluorescence and scatter will be evaluated. Finally, labeling and detection strategies for the simultaneous use of several probes, each labeled with a separate infrared fluorescent dye, will be developed. The fluorochromes will differ in their absorption and/or emission spectra, thus allowing multicolor detection of several probes at the same time. The simultaneous use of different dyes could increase hybridization and detection efficiency by an additional order magnitude towards, the end of this project. Integration of fluorescence detection and automatic hybridization. We will integrate infrared fluorescence scanning and automatic hybridization to eliminate all manual steps in the hybridization/detection step of multiplex sequencing. This will allow completely unattended operation of the automatic hybridizer, thereby increasing the operation time of the machines and improving efficiency. Image data will be transferred directly to processing computers, with intermediate local data storage for increased robustness and reliability. The building of integrated machines for automatic hybridization and detection will be an important step towards a fully automated system for multiplex sequencing.
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