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

Most studies investigating microbial community dynamics in different environments focus on bacteria. However, although often much less abundant in a microbial community, archaea can be of key importance. Nevertheless, the abundance of archaea is often overlooked or underestimated. A multitude of 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 DNAmock specifically designed to study archaea is available to date. Objectives: This study aims to validate different primer pairs to estimate abundances of archaea in a microbial community. Methods: We developed five mock communities containing different mixtures of DNA of six archaeal and four bacterial species. Next, we evaluated nine primer pairs targeting different regions of the 16S rRNA gene and are estimated to amplify specifically bacteria, archaea or both. Results: Much variation among the different primer pairs was observed. Although some primer pairs 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 for archaea 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 in the mock were observed, which should be eliminated further. Overall, our study shows the necessity of primer validation with a DNA mock.