New insights in host-associated microbial diversity with broad and accurate taxonomic resolution

O_LIDeep microbiome profiling has sparked a revolution in biology, recontextualizing mechanisms such as macroorganismal adaptation and evolution. Amplicon sequencing has been critical for characterization of highly diverse microbiomes, but several challenges still hinder their investigation: (1) Poor coverage of the full diversity, (2) Read depth losses and (3) Erroneous diversity inflation/distortion.\nC_LIO_LIWe developed a modular approach to quickly profile at least 8 interchangeable loci in a single sequencing run, including a simple and cost-effective way to block amplification of non-targets (like host DNA). We further correct observed distortion in amplified diversity by phylogenetically grouping erroneous OTUs, creating a phylogeny-based unit we call OPUs.\nC_LIO_LIOur approach achieves full, accurate characterization of a mixed-kingdom mock community of bacteria, fungi and oomycetes at high depth even in non-target contaminated systems. The OPU concept enables much more accurate estimations of alpha and beta diversity trends than OTUs and overcomes disagreements between studies caused by methodology. Leveraging the approach in the Arabidopsis thaliana phyllosphere, we generated to our knowledge the most complete microbiome survey to date.\nC_LIO_LIMicrobiomes are extremely diverse, extending well beyond bacteria and fungi. Our method makes new questions in a variety of fields tractable with accurate, systems-based overviews of microbial community structures.\nC_LI