16S rRNA Resolution

Do you require the entire length of the 16S rRNA sequence for identification?

For all necessary bioinformatics calculations, a complete 16S rRNA gene sequence is defined as the DNA sequence region between universal PCR primers 27F and 1492R for Bacteria (Lane, 1991), and between PCR primers A25F and U1492R for Archaea (Dojka et al., 1998). This allows the fair calculation of sequence similarity between PCR-derived and genome-derived reference sequences. The use of these particular regions in the [SaaS] 16S database was given by Kim et al. (2012); in the latter study, the determination of the 16S cutoff, i.e., 98.7% similarity, for species delineation was proposed based on this region as a full-length 16S sequence (Kim et al. 2014).

To reiterate more succinctly:

A complete 16S rRNA gene sequence is the DNA between the primers 27F and 1492R for Bacteria, and A25F and U1492R for Archaea.

The complete 16S rRNA gene sequence serves as a reference against which partial 16S rRNA gene sequences (obtained from high throughput sequencing) can be compared. Complete 16S rRNA gene lengths vary depending on species, and a complete or nearly complete sequence is generally required for taxonomic analyses.

Then how do we determine whether a 16S rRNA gene segment that was sequenced from a sample is complete or nearly complete? We use a measure called completeness.

Completeness is an objective measure of the degree of coverage of a query 16S rRNA gene sequence with respect to the full-length, complete 16S rRNA gene sequence.

Formula

Mathematically, completeness is defined as (Kim et al., 2012):

Where L is the length of a query sequence and C is the length of the most similar sequence that is regarded as complete (using the definition above). The most similar sequence in the database of complete sequences is identified by using an algorithm called USEARCH.

The suggested minimum threshold for using a 16S rRNA gene sequence for taxonomic purposes is 95% completeness, as incomplete or partial sequences with low completeness scores will have insufficient resolving power, resulting in erroneous identification results.

Example

Consider a partial 16S rRNA gene sequence from the strain Nocardia carnea that’s 606 bp in length (Accession AY756546.1, 606 bp):

Completeness is 42.1% because the query 16S rRNA sequence (indicated in blue) only spans from 19~625 bp of the complete 16S rRNA sequence (indicated in red), which is 1439 bp long.

References

Dojka, M. A., Hugenholtz, P., Haack, S. K., & Pace, N. R. (1998). Microbial diversity in a hydrocarbon-and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Applied and Environmental Microbiology, 64(10), 3869-3877. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., … & Chun, J. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. International journal of systematic and evolutionary microbiology, 62(Pt_3), 716-721.

Kim, M., Oh, H. S., Park, S. C., & Chun, J. (2014). Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International journal of systematic and evolutionary microbiology, 64(Pt_2), 346-351.

Lane, D. J. (1991). 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics.