Abstract
Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.
Highlights
Over the past few decades, the 16S rRNA gene has been generally accepted as a standard for identification and classification of prokaryotic species owing to its structure, containing conserved and variable regions, and occurrence in all organisms
Information on variable 16S rRNA gene copies aid in bacterial species identification we investigated highly similar multiple copies of 16S rRNA gene sequences of different strains of E. coli and species of Shigella, as well as Bacillus thuringiensis and Bacillus cereus
Variation of the copies may arise from mutations and, classified by Sun et al [21] into five categories: intervening sequence (IVS), inserts larger than 10 nucleotides, deletion, truncation, and regional diversity or random
Summary
Over the past few decades, the 16S rRNA gene has been generally accepted as a standard for identification and classification of prokaryotic species owing to its structure, containing conserved and variable regions, and occurrence in all organisms. Information on variable 16S rRNA gene copies aid in bacterial species identification sequence information, the limited resolving power of 16S rRNA gene sequences has become obvious, especially when closely related organisms are being inspected [2]. Since the 16S rRNA gene has been used as a standard for classification, presence of multiple copies with sequence variations may pose an obstacle in the classification [3]. With the introduction of a number of novel and affordable next-generation sequencing technologies, the cost of genome sequencing has decreased rapidly and the clone-based Sanger sequencing of 16S rRNA gene has largely been replaced by various platforms such as 454/Roche (454 Life Sciences, Branford, CT), Illumina (Illumina, Inc., San Diego, CA), and Ion Torrent platforms (Ion Torrent Systems, Inc., Gilford, NH) [5,6,7]
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