Gigapixel digital PCR in Giant Unilamellar Vesicles

摘要 传染病仍然是地球仪发病的主要原因。成功的医疗干预 需要准确的诊断，这可能是具有挑战性的，因为感染之间的症状共享。此外，委员会认为， 日益增加的耐药性使得鉴定和表征病原微生物并选择 及时有效的治疗。在这次竞争性的更新中，我们将扩展在 我们以前的R 01，使快速，灵敏，信息丰富的传染病诊断。我们的新 技术，我们称之为千兆像素NGS，扩展了我们的千兆像素数字PCR（dPCR）技术， 通过整合强大的下一代测序功能，核心创新 Gigapixel dPCR是在双重乳液囊泡中进行dPCR测定，而不是传统的水包水法。 商业dPCR仪器（Bio-Rad，RainDance）使用的油滴。囊泡满足了 专用液滴分析仪，并允许普通流式细胞仪用于定量。流式细胞仪 在研究和临床实验室中无处不在，具有上级分析能力（即速度、灵敏度和 多路复用）。这允许千兆像素dPCR增加 可分析的隔室数量超过1亿个，灵敏度提高100倍 量化。此外，流式细胞仪可以分选出PCR检测到DNA靶点的囊泡， 在现有的液滴分析仪中没有发现的功能。 在这次竞争性更新中，我们将利用千兆像素NGS的检测、分离和测序能力， 感染性病原体基因组直接从患者样本。拟议的工作流程将提高效率 和病原体测序的灵敏度，以低得多的成本产生更高质量的基因组草案。合作 与加州大学旧金山分校的传染病医生Charles Chiu博士合作，专门从事临床样本测序， 病原体检测，我们将开发生物信息学工具来询问恢复的基因组， 生物标志物序列，如毒力因子和耐药基因。通过进一步与 Fluent Biosciences，我们将使Gigapixel NGS适应其商业平台， 由研究和临床实验室作为市售试剂盒实施。 我们建议的具体目标是： 1)将Gigapixel PCR以Gigapixel NGS的形式扩展到临床相关平台。 2)展示细菌感染的综合定量和准确的基因组测序， 脑膜炎 3)应用计算机技术从（液体）活检中鉴定和诊断黄病毒感染 例如血浆和脑脊液。

ABSTRACT Infectious disease remains a major cause of morbidity across the globe. Successful medical intervention requires accurate diagnosis, which can be challenging due to symptoms shared among infections. Moreover, increasing drug resistance makes it important to identify and characterize pathogenic microbes and choose effective treatments in a timely manner. In this competing renewal, we will extend technology developed under our previous R01 to enable rapid, sensitive, and information-rich infectious disease diagnosis. Our new technology, which we dub Gigapixel NGS, extends our Gigapixel digital PCR (dPCR) technology from the previous R01 by incorporating a powerful next-generation sequencing capability. The core innovation of Gigapixel dPCR was to perform dPCR assays in double emulsion vesicles, rather than conventional water-in- oil droplets utilized by commercial dPCR instruments (Bio-Rad, RainDance). Vesicles obviate the need for specialized droplet analyzers and allow common flow cytometers to be used for quantification. Flow cytometers are ubiquitous in research and clinical labs, with superior analysis capabilities (i.e. speed, sensitivity, and multiplexing) in comparison to specialized droplet analyzers. This allows Gigapixel dPCR to increase the number of compartments that can be analyzed to over 100 million, providing a 100-fold increase in sensitivity of quantification. Moreover, flow cytometers can sort out vesicles in which PCR has detected a DNA target, a functionality not found in existing droplet analyzers. In this competing renewal, we will leverage the capability of Gigapixel NGS to detect, isolate and sequence infectious pathogen genomes directly from patient samples. The proposed workflow will increase the efficiency and sensitivity of pathogen sequencing, yielding higher-quality draft genomes at far lower cost. In collaboration with Dr. Charles Chiu, an infectious disease physician at UCSF specializing in clinical sample sequencing for pathogen detection, we will develop bioinformatic tools to interrogate the recovered genomes for relevant biomarker sequences, such as virulence factors and drug resistance genes. Through further collaboration with Fluent Biosciences, we will adapt Gigapixel NGS to their commercial platform, allowing the protocol to be implemented by research and clinical labs as a commercially available kit. The Specific Aims of our proposal are to: 1) Extend Gigapixel PCR into a clinically relevant platform, in the form of Gigapixel NGS. 2) Demonstrate integrated quantification and accurate genome sequencing of bacterial infections in meningitis. 3) Apply the technology for in silico identification and diagnosis of flavivirus infections from (liquid) biopsy samples such as plasma and cerebrospinal fluid.