Taxonomic resolution of the ribosomal RNA operon in bacteria: implications for its use with long-read sequencing

DNA barcoding through the use of amplified regions of the ribosomal operon, such as the 16S gene, is a routine method to gain an overview of the microbial taxonomic diversity within a sample without the need to isolate and culture the microbes present. However, bacterial cells usually have multiple copies of this ribosomal operon, and choosing the 'wrong' copy could provide a misleading species classification. While this presents less of a problem for well-characterized organisms with large sequence databases to interrogate, it is a significant challenge for lesser known organisms with unknown copy number and diversity. Using the entire length of the ribosomal operon, which encompasses the 16S, 23S, 5S and internal transcribed spacer regions, should provide greater taxonomic resolution but has not been well explored. Here, we use publicly available reference genomes and explore the theoretical boundaries when using concatenated genes and the full-length ribosomal operons, which has been made possible by the development and uptake of long-read sequencing technologies. We quantify the issues of both copy choice and operon length in a phylogenetic context to demonstrate that longer regions improve the phylogenetic signal while maintaining taxonomic accuracy.