Enhanced specificity ofBacillusmetataxonomics using atuf-targeted amplicon sequencing approach

Abstract

AbstractBacillusspecies are ubiquitous in nature and have tremendous application potential in agriculture, medicine, and industry. However, the individual species of this genus vary widely in both ecological niches and functional phenotypes, which, hence, requires accurate classification of these bacteria when selecting them for specific purposes. Although analysis of the 16S gene has been widely used to disseminate the taxonomy of most bacterial species, this gene fails proper classification ofBacillusspecies. To circumvent this restriction, we designed novel primers and optimized them to allow exact species resolution ofBacillusspecies in both synthetic and natural communities using high-throughput amplicon sequencing. The primers designed for thetufgene were not only specific for theBacillusgenus but also sufficiently discriminated species bothin silicoandin vitroin a mixture of 11 distinctBacillusspecies. Investigating the primers using a natural soil sample, 13 dominant species were detected includingBacillus badius,Bacillus velezensis, andBacillus mycoidesas primary members, neither of which could be distinguished with 16S sequencing. In conclusion, a set of high-throughput primers were developed which allows unprecedented species-level identification ofBacillusspecies, including agriculturally important species.