(732h) Isolation and Metagenome Analysis of Single Microdroplet-Cultivated Microbes

Droplet microfluidics is a powerful tool for investigating biological systems including microbial communities, featuring high throughput and small material requirement. However, isolation and analysis of single droplets become very challenging after generation and incubation of these mini-bioreactors. In particular, the amount of material, such as DNA, from droplets is often not enough for analytical techniques such as sequencing. In this work, we have developed a microfluidic device utilizing a pneumatic closed valve to space and dispense single droplets from a large pool. We achieved up to 83% accuracy in dispensing a single droplet. After droplet dispensing, we successfully amplified the genomic DNA from E. coli cells cultured in a single droplet via Multiple Displacement Amplification (MDA). We verified the fidelity of the amplified genome by RT-PCR and whole genome sequencing. We also amplified DNA from droplets containing two species of bacteria and used RT-PCR to confirm both genomes were amplified by MDA. Our technology platform is capable of isolating single droplets for downstream DNA amplification and analysis, widely applicable to the investigation of numerous microbial communities. Applying this technology to a real microbiome, our ongoing work is examining microbial interactions in the human gut by droplet co-cultivation, MDA, and metagenomic sequencing.