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Appl Environ Microbiol. 2019 Feb 1. pii: AEM.02659-18. doi: 10.1128/AEM.02659-18. [Epub ahead of print]

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

Author information

Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Chascomús, Buenos Aires, Argentina.
Department of Marine Biology and Oceanography, Institut de Ciències del Mar, CSIC, Barcelona, Catalunya, Spain.
Ocean Sciences Department, University of California, Santa Cruz (UCSC), CA, USA.
Centro Oceanográfico de Málaga, Instituto Español de Oceanografía, Fuengirola, Málaga, Spain.
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-CONICET, Chascomús, Buenos Aires, Argentina.
Centre for Marine Ecosystems Research, School of Science, Edith Cowan University, Joondalup, WA, Australia.


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 picocyanobacterial 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 to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE: The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5-V7 of the 16S rRNA gene are better suited to study the diversity, community structure and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S RNA gene sequence. Further, we successfully designed a new set of primers flanking the V5-V7 regions whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.


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