Analysis of V3V4 16s rRNA primer systems for profiling thermophilic microbial communities

The goal of our study was a comparative analysis of microbial communities in Kamchatka hot springs. For the most adequate characterization of these communities we analysed published 16S rRNA gene primers in order to evaluate their ability to amplify such genes of the representatives of deeply branching lineages including thermophilic microorganisms. First, we analyzed 125 unique sequences of declared to be universal for Bacteria, Archaea or both 16S rRNA gene primers and found out that the V3-V4 16S rRNA gene region corresponds best to our task. Next, we analyzed 16 forward and 15 reverse published primer sequences for this region in silico and found that in all cases the use of these primers can lead to the elimination of important thermophilic lineages, especially of deeply branching archaeal lineages. By aligning primer sequences aiming the same conservative regions, we obtained sequences with the maximum number of degenerations after which the usefulness of each individual degeneration was consistently assessed. As a result, we designed primer system for the V3-V4 16S rRNA gene region which can be considered as an optimal one for the analysis of complex thermophilic microbial communities. In vitro this primer system was tested in comparison with widely used primer systems for 16S rRNA microbial community analysis on the Illumina MiSeq platform. It was found that the use of our primers resulted in a significant increase in the overall Shannon index and of archaeal diversity in Kamchatka hot springs. In particular, we found a high abundance and diversity of Nanoarchaeota, Aigarchaeota, Bathyarchaeota, uncultured Thermoplasmatales and other archaeal lineages, content of which in many hot springs exceeded 50%.