Coupling FACS and genomic methods for the characterization of uncultivated symbionts

Symbioses between microbes are likely widespread and functionally relevant in diverse biological systems; however, they are difficult to discover. Most microbes remain uncultivated, symbioses can be relatively rare or dynamic, and intercellular connections can be delicate. Thus, traditional methods such as microscopy are inadequate for efficient discovery and precise characterization of novel interactions, their metabolic basis, and the species involved. High-throughput metagenomic sequencing of entire microbial communities has revolutionized the field of microbial ecology; however, genomic signals from symbionts can get buried in sequences from abundant organisms and evidence for direct links between microbial species cannot be gained from bulk samples. Thus, a specialized approach to the characterization of symbioses between naturally occurring microbes is required. This chapter presents methods for combining fluorescence-activated cell sorting to isolate and separate uncultivated symbionts with molecular biology techniques for DNA amplification in order to characterize uncultivated symbionts through genomic and metagenomic techniques.