Massively Parallel Single-Cell Sequencing of Genetic Loci in Diverse Microbial Populations

Single cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology. However, we lack a broadly applicable and accessible method to study this heterogeneity at the single cell level. Here, we introduce a simple, robust, and generalizable platform for quantitative and massively parallel single cell sequencing of target genetic loci in microbes using ultrahigh-throughput droplet microfluidics (Droplet Targeted Amplicon Sequencing or DoTA-seq). Using DoTA-seq, we elucidate the highly diverse single cell ON/OFF states of the phase-variable capsule synthesis operons in the prevalent human gut species Bacteroides fragilis . In addition, we quantify the shifts in antibiotic resistance gene abundances in different species in a 25 member human gut microbial community in response to antibiotics. By sequencing tens of thousands of single-cells derived from a human fecal sample, we identify links between plasmid replicons and the taxonomic lineages of their associated hosts. In sum, DoTA-seq is an accessible and broadly applicable tool for profiling single-cell genetic variation in microbiomes.### Competing Interest StatementF.L. and O.S.V. have filed a provisional patent: Methods for isolating and barcoding nucleic acid. US Provisional Application 63/337468.