A Comparison of Primers in 16S rRNA Gene Surveys of Bacteria and Archaea from Volcanic Caves

Abstract

The construction of amplified 16S rRNA gene libraries has been a major methodology for bacterial/archaeal environmental community profiling for decades. Over the years, a variety of alternative primer sets targeting different portions of the rRNA gene have been used. Gradually a widespread collaborative effort, supported by the Earth Microbiome Project (EMP) and other community initiatives, has settled on primers targeting the V4 region (515F/806R) of the 16S rRNA gene, which amplifies both Archaea and Bacteria. Understudied volcanic cave ecosystems possess a high proportion of unclassified bacterial and archaeal lineages, highlighting the selection of optimal primers for community analysis in this environment. Therefore, we investigated the coverage of EMP and alternative archaea-specific (519F/1017R) and bacteria-specific (27F/515R) primers, using microbial and mineral deposits from two volcanic caves. In silico and sequencing analyses showed that the two bacterial primers captured similar amounts and types of Bacteria, while significant differences in the type and abundance of taxa detected between the EMP primers and archaea-specific primers were noted. Our results validate the use of V4 EMP primers for Bacteria in this cave ecosystem, but strongly suggest that Archaea diversity is better captured with the archaea-specific primer in the cave deposits studied.