For our purposes, a complete 16S rRNA gene sequence is defined as the DNA sequence region between PCR primers 27F and 1492R for Bacteria (Lane, 1991), and between PCR primers A25F and U1492R for Archaea (Dojka et al. , 1998). This definition is based on the most widely used primers in16S rRNA gene sequencing.
To reiterate more succinctly:
A complete 16S rRNA gene sequence is the DNA between PCR primers 27F and 1492R for Bacteria, and between PCR primers 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.
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.
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.
- 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. Appl Environ Microbiol 64, 3869-3877.
- Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H., Yi, H., Won, S. & Chun, J. (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716-721.
- Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics. Edited by E. Stackebrandt and M. Goodfellow. Chichester: Wiley.
Last Updated 09/26 (SJK)