Droplet-Based Digital Microfluidic Genome Sequencing

描述(由申请人提供)：R21部分(第1年)的总体目标是演示如何修改现有的基于液滴的微流控电润湿技术，以通过合成反应化学进行测序，其基础是将克隆在微珠上的DNA引入液滴，该液滴经历重复的核苷酸添加和洗涤循环。其目的是：1)采用电润湿技术展示基于珠子的液滴形式的合成反应化学，包括珠子洗涤、珠子滞留和酶副产物向远程检测地点的传输；2)展示扩散反应方程的定量模型，描述液滴中微珠的序列和随后产生的光量；3)通过模拟演示单面组装策略将提供相当于鼠标的组装。R33部分的目标是为第一次基因组测序示范(第4-5年)开发一种综合检测战略(第2-4年)。主要目标是：1)采用电润湿技术，在2-5年内提供实验的、集成的平台，以支持珠基液滴形式的合成反应化学的研究，包括珠洗、保留、芯片上分配以及利用集成的CMOS光传感器阵列将酶副产物传输到远程阵列；2)证明电润湿技术可以扩展到皮升液滴形式，包括具有2%体积控制的芯片上分配、每个分配液滴的可重复珠数、洗涤操作中100%的珠子保留率、珠滴和洗涤液滴的受控合并和分离，以及合成测序过程的良好重复性和可控性(2-5年)；3)通过合成实验确定液滴测序中的读取长度限制，并实施软件和信号处理策略和组装方法以改善读取长度和数据质量，目标是在第4年之前演示1,000至10,000个碱基对读取；在4-5年内对小基因组进行测序。

DESCRIPTION (provided by applicant): 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.