Droplet-Based Digital Microfluidic Genome Sequencing

描述（由申请人提供）：R21部分（第1年）的总体目标是证明如何修改现有的基于液滴的微流体电润湿技术，以通过合成反应化学进行测序，该合成反应化学基于将克隆在微珠上的DNA引入到经受核苷酸添加和洗涤的重复循环的液滴中。其目标是：1）调整电润湿技术以展示基于珠的液滴形式的合成反应化学，包括珠洗涤、珠保留和酶副产物到远程检测位点的运输; 2）展示描述液滴中的微珠上的测序和随后产生的光量的扩散反应方程的定量模型; 3）通过模拟证明单侧组装策略将提供与Mouse等效的组装。R33部分的目标是为第一个基因组测序示范（第4-5年）开发一个综合检测策略（第2-4年）。主要目标是：1）在第2-5年采用电润湿技术提供实验性的集成平台，以支持基于珠滴形式的合成反应化学研究，包括珠洗涤、保留、芯片上分配以及将酶副产物输送到具有集成CMOS光电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，包括芯片上分配，2%体积控制，每个分配液滴的珠粒数量可再现，洗涤操作期间100%珠粒保留，珠粒液滴和洗涤液滴的受控合并和分裂，以及合成测序过程的良好再现性和控制（2-5年级）; 3）通过实验确定基于液滴的合成测序中的读段长度限制，并实施软件和信号处理策略和组装方法，以提高读取长度和数据质量，目标是在第四年展示1，000至10，000个碱基对读取;在4-5年级完成一个小的基因组测序。

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.