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

描述（由申请人提供）：尽管我们在当前的R21提案中已经在证明数字微流体在合成测序中的能力方面取得了重大进展，但该技术应用于实现长读取的一些关键实验方面需要证明。因此，第一年的总体目标是通过结合以下改进来延长基于液滴合成测序的焦磷酸测序反应化学的阅读长度：1)将DNA固相附着与重复的液滴洗涤和暴露于试剂液滴相兼容；2)在硅油介质中以液滴形式演示合成反应化学；3)将电润湿芯片与片外试剂供应源连接，实现不间断的350碱基对读取。本提案第2年和第3年的具体目标是：1)使用基于检测到的光信号反馈控制的自适应试剂递送策略确定读取长度和吞吐量限制。证明均聚物区域可以通过没有或可忽略不计的后续序列精度的退化进行测序；2)扩展模拟能力，开发基于物理的模型来估计可实现的精度，并利用这些见解来增加基于液滴的焦磷酸测序的读取长度；3)制定平行反应的策略和架构，并对电润湿芯片面积、CMOS光电探测器阵列尺寸和电极尺寸进行估算，以实现我们10年内10000个平行反应的目标；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)通过实验确定基于合成的液滴测序的读取长度限制，并实施软件和信号处理策略来提高读取长度，提高数据质量和吞吐量，目标是到第三年实现1,000到10,000个碱基对的读取。研究设计是基于可验证的子任务，每个目标是由小组领导驱动。我们提出的改进的焦磷酸测序方法的两个关键发明是：1)分离测序和检测步骤，使用反馈在遇到和检测到均聚物区域的任何DNA位点分别添加额外的核苷酸；2)利用基于液滴的电润湿技术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.