Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

Microbial communities often respond to various challenges, such as the presence of antibiotics, as a whole. Dissecting these community-level effects into separate acting entities requires the identification of organisms that carry functional genes for the observed feature. However, unculturable microbes abound in various environments, hence making the identification challenging. Here we present a cultivation-free technique that can be utilized to link functional genes with carrying bacterial species at single-cell resolution. The developed protocol is relatively simple to use, utilizes commercially available droplet microfluidics devices, does not require toxic reagents (as compared to some previous methods), eliminates invalid signals emerging from extracellular DNA, and allows simultaneous analysis of community diversity via 16S rRNA gene sequencing. The method can be customized for any given genetic trait to accurately identify its hosting subpopulation from a heterogeneous and potentially uncultivable bacterial community. ### Competing Interest Statement The authors have declared no competing interest.