Tuesday, May 13, 2025
5/13/2025
ANALYZING MULTIPLEXED AMPLICONS FOR ADVANCED SEQUENCING - Although the sequence of the human genome was substantially completed in 2002, there is still a huge need for the production of DMA sequence data. The number of organisms and individuals which may be sequenced is still significantly constrained by the lengthy time, high complexity and exorbitant costs of today's Sanger sequencing methods. Significant effort is currently going into the creation of new chemistries to read out the bases of DMA strands without the use of electrophoresis; however, the sample preparation processes to feed these sequencing systems has yet to be fully worked out and optimized. Many of the advanced sequencing methods require many millions of sets of amplified DNA fragments, with each set being comprised of millions of identical fragments. These sets are attached to a solid support which is then processed to read out the sequence. We propose the use of novel ultra-high-density micromachined plates as the vessels for preparing nanoliter-scale reactions for sequencing. This preparation generally includes DNA fragmentation, amplification, enrichment and solid-support-attachment steps which result in a chip with a large number of samples ready to be sequenced. Currently proposed sample preparation methods using polonies or beads may require a large number of steps to succeed and researchers may not have the freedom to practice certain protected bead-based technologies. The proposed Phase I project will include the demonstration of DNA amplification of several fragment species and a determination of how many different fragments may be reasonably analyzed from a single spot of an advanced sequencing by synthesis system. The ability to analyze multiple species in each spot could improve throughput and significantly simplify-sample preparation steps. Successful accomplishment of the Phase I milestones and completion of a subsequent Phase II and Phase III projects will result in the development of an ultra-high throughput prototype system which can produce cost-effective high-density chips for use in conjunction with advanced DNA sequencing instruments. Ultimately, this next generation of sample preparation and sequencing technologies, which can produce DNA sequence several orders of magnitude cheaper and faster than existing systems, can help to make the promise of individualized therapeutics a reality.
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