Abstract
Due to their fundamentally different biology, archaea are consistently overlooked in conventional microbiome surveys. Using amplicon sequencing, we evaluated methodological set-ups to detect archaea in samples from five different body sites: respiratory tract (nasal cavity), digestive tract (mouth, appendix, and stool) and skin. With optimized protocols, the detection of archaeal ribosomal sequence variants (RSVs) was increased from one (found in currently used, so-called “universal” approach) to 81 RSVs in a representative sample set. The results from this extensive primer-evaluation led to the identification of the primer pair combination 344f-1041R/519F-806R which performed superior for the analysis of the archaeome of gastrointestinal tract, oral cavity and skin. The proposed protocol might not only prove useful for analyzing the human archaeome in more detail but could also be used for other holobiont samples.
Highlights
The importance of microbial communities to human and environmental health motivates microbiome research to uncover their diversity and function
Primer pairs were evaluated with respect to the following characteristics: high in silico specificity for archaeal 16S rRNA genes, amplicon length of 150 to 300 bp, and in vitro capability to amplify diverse archaeal 16S rRNA genes from a variety of human specimens
Besides archaea-specific primer pairs, two widely used “universal” primers (Caporaso et al, 2012; Walters et al, 2016), namely 515F-806uR and 515FB-806RB, were evaluated as well to assess the potential of “universal” primers to display archaeal diversity associated with the human body
Summary
The importance of microbial communities to human and environmental health motivates microbiome research to uncover their diversity and function. For better comparability among different studies, standard operational procedures for sampling, storing samples, DNA extraction, amplification and analysis were set-up [e.g., the Earth Microbiome Project (Gilbert et al, 2014) and the Human Microbiome Project (Methé et al, 2012)]. This includes the use of so-called “universal primers” (Caporaso et al, 2012; Klindworth et al, 2013; Walters et al, 2016), to maximally cover the prokaryotic diversity. Most microbiome studies still overlook the human archaeome (Eisenstein, 2018; Moissl-Eichinger et al, 2018)
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