Analysis of 16S rRNA Primer Systems for Profiling of Thermophilic Microbial Communities

Thermophilic microorganisms are of special interest for phylogenetics and research in prokaryotic evolution, since many of them belong to deep branches of the tree of life. For this reason a number of broadly used universal primer systems for the 16S rRNA gene eliminate certain groups of thermophilic prokaryotes from their detection spectra. In the present work, the known 16S rRNA gene primer systems were analyzed in order to determine their ability to reveal members of deep phylogenetic lineages containing thermophilic microorganisms. It was shown that application of most of the published primer systems could result in elimination of certain groups of thermophilic prokaryotes. In silico analysis of existing primer systems was used to select the primer system for the V3‒V4 region of the 16S rRNA gene, which minimized elimination of thermophilic prokaryotic groups. Comparison of the proposed system with the previously published ones was carried out using high-throughput sequencing. Statistical analysis of the sequencing results based on the Shannon and Chao1 indexes revealed high efficiency of the proposed system for analysis of microbial communities of Kamchatka hot springs.