UC San Diego

DNA sequencing is used in a vast array of applications including pharmacogenetics, cancer research, biodiversity studies and genotyping. With an increasing demand for faster, more accessible DNA sequencers, current next- generation technologies have made astonishing advances that promise to break the $1,000 per human genome barrier. Here we present a novel DNA sequencing strategy called Natural DNA Sequencing by Synthesis (nSBS). The key aspect of nSBS is the incorporation of a small percentage of a non-terminating fluorescently labeled nucleotide along with the natural nucleotide in the cyclic nucleotide-by- nucleotide DNA synthesis process for sequence detection. The sparse incorporation and subsequent removal of the fluorescent labels minimizes the modification of the natural structure of the extending DNA template, ensuring that subsequent DNA synthesis will be mostly natural and un-perturbed. Our platform allows scaling to high-density arrays for genome sequencing due to the decoupling of the sequencing reaction from the detection step. A theoretical model of the nSBS process has been developed, utilizing Monte Carlo simulations with random sequences from the human genome to assess the feasibility of the method. The model revealed that homopolymer stretches up to 20 bases long can be sequenced with high accuracy and Q20 (with 99% accuracy) read lengths of up to 1,000 bases can be achieved with less than 10,000 copies of DNA templates and 10% labeled incorporation. A major challenge to the implementation of nSBS was the design and construction of an automated sequencing platform. An overview of the hardware and software of this system is given. Finally, results from the implementation of nSBS are provided beginning with a study of the kinetics of the incorporation of fluorescently labeled nucleotides. In addition, we demonstrate fractional labeling of a synthetic DNA template. By combining the best proven aspects of SBS with streamlined methods for DNA amplification and high-speed fluorescence imaging, nSBS can provide a platform for inexpensive genome resequencing and de novo sequencing