Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of organisms from complex microbial communities. The hypervariability and multi-copy nature of the small ribosomal subunit RNA (16S rRNA) gene, coupled with the availability of growing sequence databases, allow taxonomic identification (potentially to species level) and improved detection.
16S metagenomics primers with broad taxonomic coverage (“universal”) are advantageous for surveying (complex) communities, but compared to taxon-specific primers, they generate fewer sequences, per each operational taxonomic unit being sequenced. With universal primers, abundant taxa will represent a large proportion of the data, while rare taxa (possibly including pathogens, bioindicators or target species) may remain undetected, depending on the sequencing depth.
In the present study, two novel 16S rRNA primer pairs against the phylum cyanobacteria were designed for NGS technologies-based applications, in the context of water quality studies.
The novel primers were extensively tested and compared to a widely adopted universal 16S rRNA primer pair, starting from cultures and heterogeneous fresh-water samples.
Compared to universal 16S rRNA primers, in silico and experimental analyses demonstrated that the novel primers showed increased specificity for cyanobacteria and proteobacteria, allowing detection, identification and relative quantification also of toxic bloom-forming microalgae, water-quality bioindicators and pathogens.
Cyanobacteria and proteobacteria formed a larger proportion of the sequences amplified by the novel primers, as compared to the universal 16S rRNA primer set (95.3 % and 94.7 % vs. 48.7 %, respectively).
The increased selectivity for cyanobacterial and proteobacterial DNA of the novel primers allows parallel sequencing of more samples, yet guarantying the same level of coverage and species discrimination for these target groups.