Comprehensive ecosystem-specific 16S rRNA gene databases with automated taxonomy assignment (AutoTax) provide species-level resolution in microbial ecology

High-throughput 16S rRNA gene amplicon sequencing is an indispensable method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases, and by the lack of a systematic and comprehensive taxonomic classification for most environmental bacteria. Here we combine high-quality and high-throughput full-length 16S rRNA gene sequencing with a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) to create robust, near-complete 16S rRNA gene databases for complex environmental ecosystems. To demonstrate the benefit of the approach, we created an ecosystem-specific database for wastewater treatment systems and anaerobic digesters. The novel approach allows consistent species-level classification of 16S rRNA amplicons sequence variants and the design of highly specific oligonucleotide probes for fluorescence in situ hybridization, which can reveal in situ properties of microbes at unprecedented taxonomic resolution.