Continuous Sequencing-By-Synthesis Based on a Digital Microfluidic Platform

描述（由申请人提供）：虽然我们在我们目前的R21提案中证明了数字微流体在合成测序中的能力方面取得了重大进展，但需要证明该技术应用于实现长读段的一些关键实验方面。因此，第一年的首要目标是通过结合以下改进来延长基于液滴的合成测序焦磷酸测序反应化学的读取长度：1）整合与重复液滴清洗和暴露于试剂液滴兼容的DNA固相附着; 2）以浸入硅油介质中的液滴形式展示合成反应化学; 3）电润湿芯片与芯片外试剂供应源的接口，以实现不间断的350个碱基对读取。本提案第2年和第3年的具体目标是：1）使用自适应试剂递送策略确定读取长度和通量限制，并基于检测到的光信号进行反馈控制。2）扩展模拟能力以开发基于物理的模型以估计可实现的准确度并使用见解来增加基于液滴的焦磷酸测序的读取长度; 3）开发用于并行反应的策略和架构，并形成对电润湿芯片面积、CMOS光电检测器阵列尺寸和电极尺寸需要缩放到我们的10年目标10，000个平行反应; 4）证明电润湿技术可以通过缩放系统尺寸来缩放到皮升液滴格式以实现高度平行的反应; 5）通过合成实验确定基于液滴的测序中的读段长度限制，并实施软件和信号处理策略，以提高读取长度、数据质量和吞吐量，目标是在第3年前实现1，000至10，000个碱基对的读取。研究设计是基于可验证的子任务，为每个目标是由小组领导人。两个关键发明是我们提出的改进的焦磷酸测序方法的基础，以获得长读段：1）将测序和检测步骤解耦，使用反馈在遇到和检测到均聚物区域的任何DNA位点处分别添加额外的核苷酸; 2）利用基于液滴的电润湿来处理非常复杂的流体处理问题。

DESCRIPTION (provided by applicant): Whereas we have made significant progress in demonstrating the power of digital microfluidics in sequencing by synthesis in our current R21 proposal, some key experimental aspects of the technology as applied to achieving long reads need to be demonstrated. Thus, the overarching aim of Year 1 is to extend the read length of the droplet based sequencing-by-synthesis pyrosequencing reaction chemistry by incorporating the following improvements: 1) integrate solid-phase attachment of DNA compatible with repeated droplet washing and exposure to reagent droplets; 2) demonstrate synthesis reaction chemistry in a droplet format immersed in a silicon oil medium; 3) interfacing of the electrowetting chip with off-chip reagent supply sources to achieve uninterrupted 350 base pair reads. Our specific aims for Years 2 and 3 of this proposal are: 1) determine read length and throughput limitations using adaptive reagent delivery strategies with feedback control based on the detected light signal. Demonstrate that homopolymer regions can be sequenced through with no or negligible degradation of the subsequent sequence accuracy; 2) extend the simulation capability to develop a physically based model to estimate the achievable accuracy and use the insights to increase the read length of droplet-based pyrosequencing; 3) develop a strategy and architecture for parallel reactions and form estimates for the electrowetting chip area, CMOS photodetector array size, and electrode size needed to scale to our 10 year goal of 10,000 parallel reactions; 4) demonstrate that electrowetting technology can be scaled to a picoliter droplet format by scaling system dimensions to achieve highly parallel reactions; 5) experimentally determine read length limitations in droplet-based sequencing by synthesis, and implement software and signal processing strategies to improve read lengths and data quality and throughput, with a goal to demonstrate 1,000 to 10,000 base pair reads by Year 3. The research design is based on verifiable subtasks for each aim that are driven by group leaders. Two key inventions underlie our proposed modified pyrosequencing approach to obtain long reads: 1) decouple the sequencing and detection steps use feedback to add separately extra nucleotides at any DNA site where homopolymer regions are encountered and detected; 2) utilize droplet-based electrowetting to handle the massively complicated fluid handling problem.