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

基于数字微流控平台的连续合成测序

• 批准号: 7324437
• 负责人: RICHARD Barton FAIR
• 金额: $ 42.24万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7324437
• 关键词: Advanced Development; Affect; Architecture; Area; Base Pairing; Base Sequence; Biochemistry; Biological Assay; Cells; Chemistry; Compatible; Computer software; DNA; Data Quality; Detection; Dimensions; Diphosphates; Distant; Electrodes; Exposure to; Feedback; Genomics; Goals; Growth; Interruption; Length; Light; Liquid substance; Methodology; Methods; Microfluidics; Microspheres; Modeling; Modification; Nucleotides; Oils; Operative Surgical Procedures; Outcome; Pathway interactions; Performance; Phase; Positioning Attribute; Procedures; Productivity; Reaction; Reading; Reagent; Research; Research Design; Research Personnel; Running; Signal Transduction; Silicon; Site; Solid; Source; Stretching; System; Technology; Testing; base; computerized data processing; cost; design; detector; digital; improved; insight; natural flow; programs; prototype; simulation; size

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.

描述（由申请人提供）：虽然我们在当前的 R21 提案中在展示数字微流体在边合成边测序中的威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取的一些关键实验方面。因此，第一年的总体目标是通过结合以下改进来扩展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演示合成反应化学； 3) 电润湿芯片与片外试剂供应源的接口，以实现不间断的 350 个碱基对读取。我们在该提案第 2 年和第 3 年的具体目标是：1）使用自适应试剂输送策略和基于检测到的光信号的反馈控制来确定读取长度和吞吐量限制。证明均聚物区域可以进行测序，而后续序列准确性不会降低或可以忽略不计； 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) 制定并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 10,000 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缩放至皮升液滴格式，以实现高度并行的反应； 5) 通过合成实验确定基于液滴的测序中的读长限制，并实施软件和信号处理策略来提高读长、数据质量和吞吐量，目标是到第 3 年展示 1,000 至 10,000 个碱基对读长。研究设计基于由小组领导者推动的每个目标的可验证子任务。我们提出的改进焦磷酸测序方法获得长读长的基础是两个关键发明：1）分离测序和检测步骤，使用反馈在遇到和检测到同聚物区域的任何DNA位点单独添加额外的核苷酸； 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。