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.

通过 16S rRNA 靶向下一代测序 (NGS) 进行的丰度分析是表征居住环境中原核种群混合物的常用程序。根据所寻址的可变区/s，由于其信息内容不同，可以获得不同的映射。在这项工作中，我们专注于废水微生物群落，并比较了几个最近开发的通用引物，这些引物针对 V1-V3、V3-V4 和 V4 区域。它们都被证明在广泛的微生物门中具有良好的性能，但浮霉菌门并未得到最佳覆盖，尤其是对于 Brocadiales 家族的成员。这种细菌是基于厌氧氨氧化工艺的新型脱氮策略的基础。为了克服这一限制，我们进行了广泛的生物信息学分析，允许设计一种引物 (Pro341FB)，与先前提出的 Pro341F 引物相比，该引物显示出对该特定门的敏感性增加。使用 16S NGS 调查对来自不同废水处理厂（活性污泥系统、厌氧消化器、好氧和厌氧颗粒）的微生物群落进行验证后，我们证明 Pro341FB 能够揭示 Candidatus Brocadiales 家族成员（加上许多其他先前未充分覆盖的原核生物）而不是 Pro341F，而不会影响其先前出色的覆盖范围。