High-resolution profiling of bacterial and fungal communities using pangenome-informed taxon-specific long-read amplicons

Abstract

High-throughput sequencing technologies have greatly advanced our understanding of microbiomes, but resolving microbial communities at species and strain levels remains challenging. Here, we developed and validated a pipeline for designing, multiplexing, and sequencing highly polymorphic taxon-specific long-read amplicons. We focused on the wheat microbiome as a proof-of-principle and demonstrate unprecedented resolution for the wheat-associated Pseudomonas microbiome and the ubiquitous fungal pathogen Zymoseptoria tritici. We achieved an order of magnitude higher phylogenetic resolution compared to existing ribosomal amplicons. The designed amplicons accurately capture species and strain diversity outperforming full-length 16S and ITS amplicons. Furthermore, we tracked microbial communities in the wheat phyllosphere across time and space to establish fine-grained species and strain-specific dynamics. To expand the utility of our approach, we generated pangenome-informed amplicon templates for additional key bacterial and fungal genera. Pangenome-informed microbiome profiling enables the tracking of microbial community dynamics in complex environments and overcomes limitations in phylogenetic resolution.