SBIR-STTR Award

Intelligent Bio-Systems is developing a high-speed, low-cost DNA sequencing-by-synthesis (SBS) system which is based on the use of cleavable fluorescent nucleotides with reversible terminator technology. The cleavable linkers utilized in these nucleotides enable efficient removal of fluorescent signals and the reversible terminator technology enables reliable sequencing of repeats. In order to extend the performance of the sequencing system, we believe it is important to minimize the effect of residual linker on the nucleotide bases left behind after cleavage. These residual linkers may disturb the DNA duplex structure and interfere with a polymerase's ability to incorporate subsequent nucleotides. The goal of this Phase I project is to synthesize and evaluate several novel cleavable nucleotides which generate minimal residues after cleavage. We anticipate that the use of such nucleotides should result in much higher cumulative cycle efficiency in the SBS and enable longer sequencing reads. During Phase I of this project we will synthesize, characterize and extensively evaluate two novel designs of nucleotides which will generate smaller residues after fluorescent signal removal. The performance of novel nucleotides will be compared with the performance of our standard design cleavable nucleotides in solution and on solid surfaces. During Phase II we will synthesize and characterize 4 nucleotides based on the best performing analogs and perform synthesis optimization and scale-up. Phase II will also involve evaluation of all 4 nucleotides in our sequencing instrument. Ultimately, the ability to produce very inexpensive detailed DNA sequence information for complex organisms' genomes will both lead to accelerated discoveries throughout biology and provide the basis for Pharmacogenomics, a new paradigm in therapeutics wherein medicines are prescribed based on individual genotypes rather than just observed symptoms.

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The objective of this proposal is to further optimize Sequencing By Synthesis (SBS) chemistry to power a highly-sensitive, low-cost, sequencing system for clinical diagnostic sequencing called PinPoint MINI. A single human exome at 30 x coverage will be sequenced on this system for less than $150 on an instrument that will sell for under $70,000. The system can run 1-20 samples in parallel generating up to 20 million reads per sample with random sample access in the instrument. Currently, there is no other system on the market which would offer comparable features and performance. The simplicity of the proposed sample preparation and the low cost will enable human exome or targeted sequencing to be widely performed in the clinic and will lead to the revolution in healthcare. This Phase II project has two overall goals: 1) improving sequencing performance (read length and quality) 2) improve sequencing speed. High quality is important to produce more reliable and failsafe genetic diagnostic information and high speed is important to deliver results to physicians and patients quickly. During Phase 2, we will optimize the speed and performance to achieve at least 150 bp read length and reduces our current cycle time to 10 minutes. In addition, we will optimize our novel SBS-walking technology to achieve at least 300 (2 x 150) bp read lengths. Dr. Jeremy Edwards's at the University of New Mexico Health Sciences Center will perform external validation for our sequencing chemistry and systems.

Public Health Relevance:
Ultimately, the ability to produce very inexpensive gene expression and detailed DNA sequence information related to cancer will lead for a much wider use of expression and sequence analyses for research and treatment of this devastating disease. In addition, the ability to analyze complex organisms' genomes at a very low cost will both lead to accelerated discoveries throughout biology and provide the basis for Pharmacogenomics, a new paradigm in therapeutics wherein medicines are prescribed based on individual genotypes rather than just observed symptoms.