Concatenated 16S rRNA Sequence Analysis Improve Bacterial Taxonomy

Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Further, advances in DNA sequencing and bioinformatics workflows facilitated sophisticated genome-based methods for microbial taxonomy. Although sequencing of 16S rRNA gene is widely employed to identify and classify the bacterial community as a cost-effective and single-gene approach. However, the accuracy of the 16S rRNA sequence-based species identification is limited by multiple copies of the gene and their higher sequence identity between closely related species. Availability of a large volume of bacterial whole-genome data provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. With defined rules, we have concatenated four 16S rRNA gene copy variants to develop a species-specific reference library. 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 of concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. The approach is very effective to classify genetically related species, and it may reduce misclassification of bacterial species and genome assemblies.