AsCoM: An ecosystem-specific reference database for increased taxonomic resolution in soil microbial profiling

Intensive agriculture systems have supported a growing human population, however,abundant use of mineral fertilizers and pesticides may negatively impact nutrient cycles and biodiversity. A sustainable alternative is to harness beneficial relationships between plants and plant-associated rhizobacteria to increase nutrient uptake and provide pathogen resistance. Plant-associated microbiota profiling can be achieved using high-throughput 16S rRNA gene amplicon sequencing. Interrogation of this data is limited by confident taxonomic classifications at high taxonomic resolution (genus- or species-level), which is not possible with the commonly applied universal reference databases. The development of high-throughput full-length 16S rRNA gene sequencing combined with automated taxonomy assignment (AutoTax) can be used to create amplicon sequence variant (ASV) resolved ecosystems-specific reference databases that can greatly outperform the traditional universal reference databases. This approach was used here to create a reference database (AsCoM) for bacteria and archaea based on 987,353 full-length 16S rRNA genes from Askov and Cologne soils. We evaluated the performance of AsCoM using amplicon data, and found that it greatly increased genus- and species-level classification compared to commonly used universal reference databases. AsCoM was utilised to evaluate the ecosystem-specific bias and resolution of amplicon primers targeting the V5-V7 region of the 16S rRNA gene that are commonly used within the plant microbiome field. Finally, we demonstrate the benefits of AsCoM through the analysis of V5-V7 amplicon data to identify new plant-associated microbes for two legume (Lotus japonicus and Medicago truncatula) and two cereal (Hordeum vulgare and Zea mays) species.