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.

Keywords:

• Archaea
• bacteria
• microbial diversity
• primers
• 16S rRNA gene
• volcanic caves

Acknowledgments

We thank the National Park Service staff at Lava Beds National Monument, in particular, Katrina Smith, David Riggs, Pat Seiser, Randall Paylor, and Dave Hays for help coordinating logistics to support our fieldwork and the Cave Research Foundation for use of their Research Center at the Monument. We thank Ara Winter and Kaitlyn Reed for their personal communication regarding the dark matter in caves. We thank William Brigg and Molly Devlin for their help with data analysis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data and supplementary materials are available at figshare: https://doi.org/10.6084/m9.figshare.12185088.v1

Additional information

Funding

This work was supported by a grant from the NASA PSTAR program [NNH16ZDA001N] (PI J. Blank). Research reported in this publication was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under award number [P30 GM110907]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.