Identification of the Archaeome by optimizing the 16S rRNA amplicon sequencing approach

In Belgium, bore-hole water samples obtained from HADES Underground Research Facility confirmed the presence of an active methanogenic microbial community. However, most studies investigating microbial community dynamics in different environments focus on bacteria. Although less abundant in a microbial community, archaea can be of key importance in deep subsurface environments and the abundance is generally overlooked or underestimated. Several studies have developed primer pairs probing to specifically amplify the archaeal 16S rRNA gene. However, none of these methods were validated with a mock community. In addition, no DNA mock specifically designed to study archaea is available to date. Aiming to validate a suitable method for the community assessment, we developed five mock communities containing different mixtures of DNA of six archaeal and four bacterial species. Thereafter, we evaluated nine primer pairs targeting different regions of the 16S rRNA gene estimated to amplify specifically either bacteria, archaea or both. Much variation among the different primer pairs was observed, although some could identify all species in the mock, the abundance of each strain could not be correctly assessed. Moreover, depending on the ratio between bacteria and archaea in the mock, several species could not be identified. Methods estimated to amplify only archaea were not specific and a low amount of reads belonging to bacteria were obtained. Specificity for archaea was obtained via a nested PCR approach. However, some biases towards specific species were observed, which should be further removed. Overall, our study shows the necessity of primer validation with a DNA mock.