The main goal of the present project is a development, implementation and testing of a highly efficient method and an instrument for DNA sequencing capable of both automated re-sequencing and de-novo sequencing of mammalian size genomes with the mouse draft quality {Nature 420:520, 2002} at 100-fold reduced cost. The proposed system will be based on highly parallel CE separation and detection of fluorescently labeled dideoxynucleotide-terminated DNA extension product generated by gel matrix-immobilized colonies of single template molecules (polonies) in two dimensional monolith multi-capillary arrays (2D-MMCA). The cost reduction will come from both using nanoliter volume reactions and employing 2D-MMCAs which increase the throughput of the CE separation and detection by at least two orders of magnitude compared to commonly used high-throughput DNA machines. We will perform the following: Development of the technology platform, building of a pilot 55x55 lane automated DNA sequencer capable of sequencing 1,000 bp/second with 450bp Q20 read length at cost of $0.007/kbp, and demonstration of the production scale re-sequencing and automated generation of a genome sequence of the quality of the mouse draft genome at less than $100,000 per mammalian size genome Further development of the proposed system and introducing the technology and the instrumentation changes enabling de-novo sequencing: Optimization of polonie's cultivation technology and obtaining an efficient amplification and cycle sequencing of 3-5kb DNA fragments; Building a pilot 100x300 lane DNA sequencer capable of automated generation of raw sequencing data at 7500 bp/second with 800 bp Q20 read length at cost of $0.002/kbp. Demonstration of the system's potential for de-novo sequencing of human size genome at 10 fold coverage for $100,000 - $200,000.
| Tsupryk, A; Tovkach, I; Gavrilov, D et al. (2008) Ultra sensitive sensor with enhanced dynamic range for high speed detection of multi-color fluorescence radiation. Biosens Bioelectron 23:1512-8 |
| Kosobokova, Olga; Gavrilov, Dmitri N; Khozikov, Vyacheslav et al. (2007) Electrokinetic injection of DNA from gel micropads: basis for coupling polony technology with CE separation. Electrophoresis 28:3890-900 |
