Development and application of droplet microfluidics for high-throughput single-cell and single-molecule genomics

基因组测序是理解生物学的一个非常强大的工具。然而，这种方法需要相对大量的DNA，通常是通过在实验室条件下培养大量的克隆细胞群获得的。对于绝大多数（>99%）抵抗培养尝试的微生物来说，这是困难的，或者根本不可能的。该提案的总体目标是开发一种新的高通量方法，用于快速鉴定和测序单个DNA分子或完整的细菌基因组，这些DNA分子或完整的细菌基因组来自大群体中的目标单细胞。通过利用基于液滴的微流体技术，其中每个亚纳升液滴相当于可以操作的反应管，将有可能显着改善当前的单细胞全基因组扩增和宏基因组学方法，同时提供令人兴奋的新可能性，例如超高通量标记和基于特定遗传标记的存在或不存在的液滴检索。 在该项目的第一步中，我们将评估选定的液滴微流体方法，通过检索和测序复杂模拟细菌群体中大肠杆菌实验室菌株的完整基因组来评估其可行性和优势。这些实验将揭示所提出的方法的优点和潜在的缺陷，并使我们能够改进我们的协议。在第二步中，我们将对尚未表征的细菌的基因组进行测序，以暴露隐藏的“微生物暗物质”，并帮助用代表性的基因组序列填充生命树。更具体地说，我们建议使用我们新的液滴微流体单细胞基因组学和宏基因组学技术，允许对水样中“稀有细菌生物圈”的至少10种丰度非常低且未培养的微生物进行基因组测序。 这项有前途的技术的发展是通过结合尖端领域的专业知识而实现的，如单细胞全基因组扩增、下一代测序、基因组学和微流体学。所提出的方法是非常原始的，并有可能成为一个非常重要的技术，基因组学研究的许多领域相对简单的技术开发工作。

Genome sequencing is an extremely powerful tool for understanding biology. However, a relatively large amount of DNA, usually obtained by growing a large clonal population of cells under laboratory conditions, is required for this approach to work. This is difficult, or simply impossible, for the vast majority (>99%) of microorganisms that resist cultivation attempts. The overall objective of this proposal is to develop a novel high-throughput method for rapidly identifying and sequencing individual DNA molecules or complete bacterial genomes from targeted single-cells of interest within a large population. By taking advantage of droplet-based microfluidics, in which every sub-nanoliter droplet is equivalent to a reaction tube that can be manipulated, it will be possible to dramatically improve current single-cell whole-genome amplification and metagenomics methods while offering exciting new possibilities such as ultra high-throughput labeling and retrieving of droplets based on the presence or absence of specific genetic markers. In the first step of this project, we will evaluate selected droplet microfluidics approaches to assess their feasibility and advantages by retrieving and sequencing complete genomes of Escherichia coli laboratory strains from a complex mock bacterial population. These experiments will reveal the strengths and potential pitfalls of the proposed methodologies and allow us to improve our protocols. In a second step, we will sequence the genomes of yet uncharacterized bacteria to expose the hidden “microbiological dark matter” and help populate the tree of life with representative genome sequences. More specifically, we propose to use our novel droplet microfluidics single-cell genomics and metagenomics technology to allow the genome sequencing of at least 10 very low-abundance and uncultured microorganisms of the “rare bacterial biosphere” in water samples. The development of this promising technology is made possible by combining expertise in cutting-edge fields such as single-cell whole genome amplification, next-generation sequencing, genomics and microfluidics. The proposed approach is highly original, and has the potential to become a very important technology for many fields of genomics research given relatively simple technology development efforts.