The overall goal of the R21 section (Year 1) is to demonstrate how existing droplet-based microfluidic electrowetting technology can be modified to perform sequencing by synthesis reaction chemistry based on the introduction of DNA cloned on microbeads into a droplet that is subjected to repeated cycles of nucleotide addition and washing.
The aims are: 1) adapt electrowetting technology to demonstrate synthesis reaction chemistry in a bead-based droplet format, including bead washing, bead retention, and transport of enzymatic by-products to remote detection sites; 2) demonstrate a quantitative model for the diffusion reaction equations that describe sequencing on microbeads in droplets and the subsequent amount of light generated; 3) demonstrate through simulation that a single-sided assembly strategy will provide an assembly equivalent to Mouse. The goal of the R33 section is to develop an integrated detection strategy (Years 2-4) for the first genome sequencing demonstration (Years 4-5). The major aims are: 1) adapt electrowetting technology to provide experimental, integrated platforms in Years 2-5 to support research in synthesis reaction chemistry in a bead-based droplet format, including bead washing, retention, on-chip dispensing, and transport of enzymatic by-products to a remote array with an integrated CMOS photosensor array; 2) demonstrate that electrowetting technology can be scaled to a picoliter droplet format, including on-chip dispensing with ? 2% volume control, reproducible numbers of beads per dispensed droplet, 100% bead retention during washing operations, controlled merging and splitting of bead droplets and wash droplets, and good reproducibility and control of sequencing-by-synthesis processes (Years 2-5); 3) experimentally determine read length limitations in droplet-based sequencing-by synthesis, and implement software and signal processing strategies and assembly methodologies to improve read lengths and data quality, with a goal to demonstrate 1,000 to 10,000 base pair reads by Year 4; sequence a small genome in Years 4-5. ? ?
