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

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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.**