Summary auto-generated
Small-subunit ribosomal RNA (SSU rRNA) sequencing has become the standard method for identifying and classifying prokaryotes. However, this study revealed unexpectedly high levels of sequence variation within prokaryotic species and strains in GenBank. Researchers examined 336 prokaryotic species with multiple SSU rRNA sequences and found that 82% of species pairs showed more variability than expected from 0.1% sequencing error rates, and 48% exceeded 1% error rates. Surprisingly, within-strain variation was similar to between-strain variation, suggesting biological sources beyond simple sequencing errors. Potential sources include interoperon differences (multiple rRNA operons per species) and strain misidentification. The variability significantly affected phylogenetic analyses and biodiversity estimates in some genera like Borrelia and Cytophaga, where sequence choice altered evolutionary relationships. The authors conclude that using single sequences from single isolates inadequately represents prokaryotic taxa and recommend sequencing multiple operons from multiple strains to obtain reliable phylogenetic hypotheses and accurate biodiversity assessments.
Key findings
- 82% of prokaryotic species pairs in GenBank showed SSU rRNA sequence variation exceeding predicted 0.1% error rates, and 48% exceeded 1% error rates
- Within-strain sequence variation was similar to between-strain variation, indicating biological causes beyond sequencing error such as interoperon differences
- In genera like Borrelia and Cytophaga, choice of SSU rRNA sequence significantly affected phylogenetic tree topology and branching patterns
- For biodiversity estimates, the overlapping ranges of interoperon (0-5%), interstrain (0-16%), and interspecific variation make it impossible to classify sequence differences without multiple sampling
- Multiple SSU rRNA sequences from multiple operons and multiple strains are necessary for reliable phylogenetic studies and accurate species identification
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Abstract
Small-subunit rRNA (SSU rRNA) sequencing is a powerful tool to detect, identify, and classify prokaryotic organisms, and there is currently an explosion of SSU rRNA sequencing in the microbiology community. We report unexpectedly high levels of intraspecific variation (within and between strains) of prokaryote SSU rRNA sequences deposited in GenBank. A total of 82% of the prokaryote species with two published SSU rRNA sequences had more variable positions than a 0.1% random sequencing error would predict, and 48% of these sequence pairs had more variable positions than predicted by a 1.0% random sequencing error. Other sources of sequence variability must account for some of this intraspecific variation. Given these results, phylogenetic studies and biodiversity estimates obtained by using prokaryotic SSU rRNA sequences cannot proceed under the assumption that rRNA sequences of single operons from single isolates adequately represent their taxa. Sequencing SSU rRNA molecules from multiple operons and multiple isolates is highly recommended to obtain meaningful phylogenetic hypotheses, as is careful attention to accurate strain identification.