A novel universal primer pair for prokaryotes with improved performances for anammox containing communities.

Abundance profiling via 16S rRNA targeted next generation sequencing (NGS) is a common procedure to characterize mixtures of prokaryotic populations inhabiting an environment. Depending on the variable region/s addressed, different maps can be obtained due to their different information content. In this work, we focussed on wastewater microbial communities and we compared several recently developed universal primers that addressed regions V1-V3, V3-V4 and V4. They all proved to have good performance over a wide range of microbial phyla, but the phylum Planctomycetes was not optimally covered, especially for members of the Brocadiales family. Such bacteria are at the basis of the novel nitrogen removal strategy based on anammox process. To overcome this limitation we performed an extensive bioinformatic analysis that allowed the design of a primer (Pro341FB) that shows increased sensitivity for this specific phylum with respect to the previously proposed Pro341F primer. Upon validation using a 16S NGS survey on microbial communities from different wastewater treatment plant (activated sludge systems, anaerobic digesters, aerobic and anaerobic granules) we demonstrated that Pro341FB is able to reveal up to 5 times more members of the Candidatus Brocadiales family (plus many other previously under-covered prokaryotes) than Pro341F, without affecting its excellent previous coverage.