Primer design for an accurate view of picocyanobacterial community structure using high-throughput sequencing

CORNEJO-CASTILLO, F.; LOGARES, R.; ACINAS, S.; FERRERA, I.; METZ, S.; GASOL, J. M.; HUBER, M. P.; SÁNCHEZ, P.; BALAGUÉ, V.; UNREIN, F.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY

AMER SOC MICROBIOLOGY

Lugar: Washington; Año: 2019 vol. 85 p. 1 - 17

0099-2240

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and so far, they have predominantly been targeted using universal bacterial primers providing a limited resolution of the picocyanobacteria community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for genera Synechococcus, Prochlorococcus and Cyanobium. An in silico analysis showed that the V5-V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zOTUs), which allowed us to detect higher genetic diversity and infer ecologically-relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door for future studies investigating picocyanobacterial diversity in aquatic systems.