Concatenated 16S rRNA sequence analysis improves bacterial taxonomy

Abstract

Background: Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Advances in DNA sequencing and bioinformatics workflows have facilitated sophisticated genome-based methods for microbial taxonomy although sequencing of the 16S rRNA gene is widely employed to identify and classify the bacterial community as a cost-effective and single-gene approach. However, the 16S rRNA sequence-based species identification accuracy is limited by multiple copies of the gene and their higher sequence identity between closely related species. The availability of a large volume of bacterial whole-genome data provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. Methods: The 16S rRNA copies were retrieved from the whole genomes in the complete stage at the Genome database. With defined rules, four 16S rRNA gene copy variants were concatenated to develop a species-specific reference library. The sequence similarity search was performed with a web-based BLAST program, and MEGA software was used to construct the phylogenetic tree. Results: Using this approach, species-specific 16S rRNA gene libraries were developed for four closely related Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae). Sequence similarity and phylogenetic analysis using concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. Conclusions: The approach is very effective in classifying genetically related species and may reduce misclassification of bacterial species and genome assemblies.