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

描述(由申请人提供)：尽管在我们目前的R21提案中，我们在展示数字微流控技术在合成测序中的力量方面取得了重大进展，但该技术应用于实现长阅读的一些关键实验方面仍需证明。因此，第一年的总体目标是通过纳入以下改进来延长基于液滴的测序-合成焦磷酸测序反应化学的读取长度：1)整合DNA的固相连接，使其与液滴的反复洗涤和暴露在试剂液滴中相兼容；2)展示浸泡在硅油介质中的液滴形式的合成反应化学；3)将电润湿芯片与芯片外试剂供应源接口，以实现不间断的350个碱基对读取。我们在本提案的第二年和第三年的具体目标是：1)使用基于检测到的光信号的反馈控制的自适应试剂传递策略来确定读取长度和吞吐量限制。证明均聚物区可以通过测序而不会或可以忽略地降低后续序列的准确性；2)扩展模拟能力以开发基于物理的模型来估计可实现的精度，并利用所获得的见解来增加基于液滴的焦磷酸测序的读取长度；3)开发并行反应的策略和架构，并形成对电润湿芯片面积、CMOS光探测器阵列大小和电极大小的估计，以达到我们10,000个并行反应的10年目标；4)证明电润湿技术可以通过缩放系统尺寸来扩展到皮升液滴格式，以实现高度平行反应；5)通过合成实验确定基于液滴的测序中的读取长度限制，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，目标是到第3年演示1,000至10,000个碱基对读取。研究设计基于由小组负责人推动的每个目标的可验证子任务。我们建议的改进焦磷酸测序方法有两项关键发明，以获得较长的读数：1)将测序和检测步骤分离使用反馈在遇到和检测到均聚物区的任何DNA位置分别添加额外的核苷酸；2)利用基于液滴的电润湿来处理极其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.