PRJNA733504
Single-cell genomics for resolution of conserved bacterial genes and mobile genetic elements of the human intestinal microbiota using flow cytometry
As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. Here, we present a method utilizing Fluorescence Activated Cell Sorting (FACS) for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA analysis, and selecting cells for metagenomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to variably provide nearly-complete coverage of bacterial genomes, as well as to permit exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae. Thus, we demonstrate an approach to flow cytometric separation and sequencing of single bacterial cells belonging to the human microbiota which has the potential to yield a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes.