Novel Primer Sets for Next Generation Sequencing-Based Analyses of Water Quality.

Elvina Lee,Maninder S Khurana,Andrew Bath,Andrea Paparini,Andrew S Whiteley,Cameron Gordon,Una M Ryan,Paul T Monis

doi:10.1371/journal.pone.0170008

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.

Highlights

The growing accessibility of generation DNA sequencing (NGS) methods has greatly advanced our understanding of microbial diversity in medical and environmental science [1,2,3,4,5]
This increased specificity for Cyanobacterial sequences was seen in the analyses performed using ProbeMatch (Table 3)
Two different primer sets targeting different regions (V3, and V6 regions) of the 16S rDNA gene were designed and tested to compare their utility. This was performed in order to overcome the limitations posed by the use of universal 16S rDNA primers that were naturally designed to amplify across as broad a range as possible of bacterial and archaeal taxa

Summary

Introduction

The growing accessibility of generation DNA sequencing (NGS) methods has greatly advanced our understanding of microbial diversity in medical and environmental science [1,2,3,4,5]. Refinement of platforms, protocols and reagents for parallel high-throughput DNA sequencing technologies allows profiling of complex microbial communities at ever-increasing resolution [6, 7]. NGS has the advantage of allowing direct analysis of communities as they exist under in situ conditions, including their genes, transcripts, proteins, and metabolites and how their reciprocal interactions impact their distribution patterns [6, 8, 9]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion