Spatial profiling of microbial communities by sequential FISH with error-robust encoding

Abstract Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we show that SEER-FISH can quantify the abundance of each taxon and map microbial biogeography on roots. SEER-FISH should enable accurate spatial profiling of the ecology and function of complex microbial communities.