The goals of this project are to develop a robust sequencing by synthesis methodology for de novo and resequencing applications using the bead-based polony technology. Our overall R & D focus is to address key aspects of the technology that need to be refined to enable robust, high quality polony sequencing. Our experience in large-scale genome sequencing will serve well to ensure that the key issues involved in optimizing the technology against current industry standards, data processing, management, and analysis are effectively addressed in a time- and cost-efficient manner.
The specific aims are to: 1) Develop effective procedures for production of paired-end PCR libraries with virtual insert sizes (distance between read pairs) in the range of 2 to 50 kilobases. 2) Develop methods for effective solid-phase template amplification on derivatized microspheres and for enrichment of beads containing amplified templates. 3) Develop methods for robust array preparation. 4) Develop procedures for fluorescent in situ sequencing by synthesis. 5) Develop an integrated data acquisition system including fluorescence microscope, automated stage, flow cell, fluidics system and control software. 6) Develop data management and assembly software. 7) Develop functional reversible chain terminators. 8) Develop modified enzymes capable of efficiently incorporating reversible terminators.
