High-Throughput Microbial Interaction Analysis through Microfluidic Droplet Microbial Co-Culture and Interaction Analysis By Barcoded Amplicon Sequencing

In the latest decade, single-cell sequencing technology has transformed the biology field. However, for the prokaryotic research field, the development of single-cell sequencing techniques has lagged far behind due to various technical challenges. Traditional methods like 16S amplicon sequencing and metagenomics lack single cell resolution and depend on complexed computational algorithms to deconvolute the data. Existing implementations of single microbe genome sequencing use complex workflows that are both labor intensive and require custom microfluid devices. These systems built and operated by well-trained researchers, precluding adoption beyond microfluidic labs. To overcome this issue, we present EASi-seq (Easily Accessible Single microbe sequencing). The EASi-seq protocol enables the accessible shotgun sequencing of individual microbes by adapting the workflow of a commercial single cell instrument originally designed for eukaryotic cell analysis. We successfully applied EASi-seq to the sequencing of a synthetic microbial consortia, a human microbiome sample, and an environmental community. Using the resulting data, we demonstrated the ability to generate detailed single microbe atlases and greatly improve the quality of concurrent metagenomic sequencing. EASi-seq achieved cost-effective single cell microbe sequencing with throughput greater than tens of thousands of cells without the need of specialized expertise with microfluidics. The ability to capture whole genome datasets from thousands of single microbes provides new opportunities for the microbiology research community.