Optimization of a microfluidic device for single bacterial cell genomics

描述(由申请人提供)：探索人类微生物组的特征是一个令人望而生畏的目标，但这一目标有望显著增强我们对健康的理解和我们对各种疾病状态的管理。在这一探索中，尤其是人类微生物区系的两个特征构成了重大挑战：尚未培育的物种的比例和数量很大，以及微生物群落的极端不平衡，导致大量潜在的重要群落成员未能在常规调查中被“看到”。识别、分离和测序单个细菌细胞的基因组的能力将使我们能够表征和了解稀有和未培养的微生物物种，并实质性地促进我们对人类微生物组的理解。在最近的工作中，已经设计和制造了一种微流控设备，其特征是模仿集成电路；该设备分离单个细菌细胞，并允许以纳升体积放大它们的基因组。在本申请中，提出了对这种微流控设备进行优化和增强的计划，以减少环境污染，更容易捕获稀有细胞类型，更快地筛选更多的细胞，并且更容易从单个细胞测量基因表达。这项工作的长期目标是加强我们对人类微生物群落的了解，特别是对新的或特征不佳的未培养微生物群落成员的了解。这项提案回应了美国国立卫生研究院人类微生物组项目提出的未得到满足的关键需求。这项建议的具体目的如下：目的1.减少环境DNA对单细胞基因组序列数据的贡献，并提高我们的细胞分选、基因组扩增微流体设备获得的序列数据的信噪比。实验方法包括将光学(激光)镊子集成到设备中。目的2.提高利用微流控装置检测和捕获稀有微生物群落成员的能力。实验方法包括将荧光原位杂交技术、特定探针和荧光成像与微流控设备相结合。目的3.利用微流控装置提高单细胞筛选分离的速度。实验方法包括更高度并行的微器件设计，激光功率和激光光路的优化，以及细胞操作的进一步自动化。目的4.提高单个细菌细胞的基因表达分析能力。该实验方法包括开发芯片上的RNA分离、反转录和使用数字聚合酶链式反应来量化单细胞转录丰度的协议。公共卫生相关性：这项研究与公共健康相关，因为它将使人们更好地了解生活在人体上和体内的微生物。因为人类固有的微生物群落有助于维持健康，当受到干扰时，会导致疾病，这项研究可能导致诊断或预测与微生物群落紊乱相关的疾病的新测试，以及维持或恢复健康的新策略。

DESCRIPTION (provided by applicant): The quest to characterize the human microbiome is a daunting goal, but one that promises to enhance significantly our understanding of health and our management of a wide variety of disease states. In this quest, two features of the human microbiota in particular, pose major challenges: the large proportion and number of as-yet uncultivated species, and the extreme unevenness of the microbial communities, with a resulting large number of potentially important community members that fail to be "seen" in routine surveys. The ability to identify, isolate, and sequence the genome of single bacterial cells would allow us to characterize and understand both rare and uncultivated microbial species, and materially advance our understanding of the human microbiome. In recent work, a microfluidic device has been designed and fabricated, with features that mimic an integrated electrical circuit; this device isolates individual bacterial cells, and allows their genome to be amplified in nanoliter volumes. In this Application, a plan is proposed for optimization and augmentation of this microfluidics device, so that environmental contamination is reduced, rare cell types are more easily captured, larger numbers of cells are screened more quickly, and gene expression is more easily measured from single cells. The long-term objectives of this work are to enhance our understanding of the human microbial communities, and in particular, of novel or poorly-characterized, uncultivated microbial community members. This proposal responds to critical unmet needs posed by the NIH Human Microbiome Project. The following are the Specific Aims of this proposal: Aim 1. To reduce the contribution of environmental DNA to single cell genomic sequence data, and increase the "signal-to-noise" ratio of the sequence data obtained with our cell- sorting, genome amplification microfluidics device. The experimental approach involves the integration of optical (laser) tweezers into the device. Aim 2. To improve the ability to detect and capture rare microbial community members with the microfluidics device. The experimental approach involves the integration of fluorescence in situ hybridization techniques, specific probes, and fluorescence imaging with the microfluidics device. Aim 3. To increase the speed of single cell selection and isolation with the microfluidics device. The experimental approach involves more highly parallel microdevice designs, optimization of laser power and laser optical path, and further automation of cell manipulations. Aim 4. To enhance the capability for gene expression analysis in single bacterial cells. The experimental approach involves the development of on-chip protocols for RNA isolation, reverse transcription, and use of digital PCR to quantify transcript abundance from single cells. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will lead to a better understanding of the microorganisms that live on and in the human body. Because the human indigenous microbial communities help to maintain health and when disturbed, contribute to disease, this research may lead to new tests for diagnosing or predicting disease associated with microbial community disturbance, and new strategies for maintaining or restoring health.