Improved detection of methanogenic archaea by validating 16S rRNA gene primers using defined archaeal–bacterial mock communities

Archaea play essential roles in global biogeochemical cycles, yet their diversity in environmental samples remains understudied. Limited coverage of universal primers towards archaea, their very low abundance relative to bacteria in the studied samples, inefficient cell lysis and the incompleteness of reference databases complicate archaeal 16S rRNA gene sequencing. Furthermore, standardization lacks due to the absence of commercially available mock communities. Here, we developed a mock community composed of mixtures of DNA from six archaeal and four bacterial strains with known genome sizes and 16S rRNA gene copy numbers. Five mock compositions with different archaeal–bacterial abundance ratios were used to evaluate two universal primer pairs and eight archaea-specific methods. The universal primer pairs detected all taxa but consistently overestimated bacterial abundance, indicating to be less suitable for environments where archaea are more dominant. Archaeal specific primer pairs showed variable specificity, with some exhibiting substantial bacterial co-amplification under certain conditions. The Arch349F–A806R primer pair demonstrated the highest archaeal specificity, the least technical variability, the most consistent agreement with expected mock compositions, and minimal artefactual amplification when applied to a complex bacterial mock community. To show its applicability on environmental samples, this method was applied on Boom Clay pore water samples, which indicated a much higher archaeal diversity than previously shown with a standard commercial method. Overall, this study showed the necessity of primer validation through mock communities and identified Arch349F–A806R as a promising primer pair for archaeal 16S rRNA gene sequencing, especially in samples containing methanogenic archaea.