Abstract
Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment.
Highlights
Generation sequencing (NGS) platforms have allowed microbiologists to gain new insights in microbial ecology [1]
One essential aspect demanding careful consideration is primer choice. Particular genes, such as the 16S rRNA gene in bacteria, contain regions that have evolved at different evolutionary rates, and as such the scientific outcome may vary with the gene region sequenced [4,5,6,7]
Because total coverage rates bias towards large bacterial phyla such as the Proteobacteria and Firmicutes, non-coverage rates were calculated per phylum (Fig. 1)
Summary
Generation sequencing (NGS) platforms have allowed microbiologists to gain new insights in microbial ecology [1]. One essential aspect demanding careful consideration is primer choice Particular genes, such as the 16S rRNA gene in bacteria, contain regions that have evolved at different evolutionary rates, and as such the scientific outcome may vary with the gene region sequenced [4,5,6,7]. The coverage rates of such primers differ with the location of their target in the 16S rRNA gene. Online matching tools such as SILVA Test Probe [12] and RDP probe match [15] have been developed to address this problem. Berry et al [16] have reported biases introduced with barcode-tagging of primers that translate into less reproducible data sets, while Wu and colleagues [17] extensively mentioned the problems of preferential amplification
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