title: Novel Primer Sets for Next Generation Sequencing-Based Analyses of Water Quality.
abstract: Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination.
authors.affiliation: Vector- and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia.
authors.forename: Elvina
authors.initials: E
authors.lastname: Lee
authors.affiliation: Vector- and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia.
authors.forename: Maninder S
authors.initials: MS
authors.lastname: Khurana
authors.affiliation: School of Earth and Environment, The University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Whiteley
authors.affiliation: Australian Water Quality Centre, South Australian Water Corporation, Adelaide, South Australia, Australia.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Monis
authors.affiliation: Drinking Water Quality Branch, Water Corporation, Leederville, Western Australia, Australia.
authors.forename: Andrew
authors.initials: A
authors.lastname: Bath
authors.affiliation: Drinking Water Quality Branch, Water Corporation, Leederville, Western Australia, Australia.
authors.forename: Cameron
authors.initials: C
authors.lastname: Gordon
authors.affiliation: Vector- and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia.
authors.forename: Una M
authors.initials: UM
authors.lastname: Ryan
authors.affiliation: Vector- and Water-Borne Pathogen Research Laboratory, School of Veterinary and Life Sciences, Murdoch University, Perth, Western Australia, Australia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Paparini
mesh_terms: D003198:Computer Simulation; D000458:Cyanobacteria / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D005618:Fresh Water / Q000382:microbiology; D057097:Harmful Algal Bloom; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010802:Phylogeny; D020560:Proteobacteria / Q000235:genetics / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D062065:Wastewater / Q000382:microbiology; D014871:Water Microbiology; D060753:Water Quality / Q000592:standards; D014881:Water Supply; D014904:Western Australia
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D062065:Wastewater