Droplet-Based Digital Microfluidic Genome Sequencing

描述（由申请人提供）：R21部分（一年级）的总体目标是展示如何通过合成反应化学将现有的基于微滴的微流控电润湿技术进行修改，从而通过将克隆在微珠上的DNA引入到经过核苷酸添加和洗涤重复循环的微滴中来进行测序。目标是：1)采用电润湿技术来演示以珠为基础的液滴形式的合成反应化学，包括珠洗涤、珠保留和酶副产物运输到远程检测点；2)展示了描述液滴中微珠的序列和随后产生的光量的扩散反应方程的定量模型；3)通过仿真证明，单面装配策略将提供相当于鼠标的装配。R33部分的目标是为第一次基因组测序示范（4-5年）制定一个综合检测策略（2-4年）。主要目标是：1)采用电润湿技术，为2-5年级的学生提供实验、集成平台，以支持基于珠状液滴形式的合成反应化学研究，包括珠状液滴洗涤、保留、片上分配和酶副产物传输到集成CMOS光敏传感器阵列的远程阵列；2)证明电润湿技术可以扩展到皮升液滴格式，包括在芯片上分配，体积控制2%，每个分配液滴的可重复数量，在洗涤操作中100%的珠滴保留率，珠滴和洗涤滴的可控合并和分裂，以及良好的可重复性和合成测序过程的控制（2-5年级）；3)通过实验确定基于液滴的合成测序的读取长度限制，并实施软件和信号处理策略以及组装方法，以提高读取长度和数据质量，目标是到第4年展示1,000到10,000个碱基对读取；在4-5年级对一个小基因组进行测序。

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.