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极其强大的工具。然而，这种方法的工作需要相对大量的DNA，通常是通过在实验室条件下培养大量克隆细胞来获得的。这是困难的，或者根本不可能的，因为绝大多数（大约99%）的微生物会抵抗培养的尝试。本提案的总体目标是开发一种新的高通量方法，用于快速识别和测序大量群体中单个DNA分子或完整细菌基因组的目标单细胞。利用基于液滴的微流体技术，每个亚纳升液滴相当于一个可操作的反应管，将有可能显著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，如超高通量标记和基于特定遗传标记是否存在的液滴检索。

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.