16S rRNA Sequencing to Identify Unknown Microbes

Need to accurately identify a new bacterium that you isolated from the environment? Ever wonder what that contaminant in your culture really is? 16S rRNA amplicon sequencing can get you there!

Overview

• Culture your bacterium and isolate DNA
• PCR using universal primers (this protocol)
• Run a portion of the reaction on an agarose gel to check the amplicon
• If needed: cut out the band and gel extract
• Clean up the DNA sample
• Submit the DNA sample for Sanger sequencing

PCR Reaction

For PCR we use universal primers U341F and UA1406R that amplify an approximately 1,000-bp (1-kbp) stretch of rDNA. They should work for nearly any bacterial or archaeal 16s rRNA sequence [1].

Using Purified gDNA as Template: It is generally best to isolate genomic DNA from your culture/cells before doing the 16S RNA PCR (using the Invitrogen PureLink kit, for example). This results in a cleaner PCR product and generally a better sequencing result. After DNA is isolated, use a Qubit to find the concentration of DNA. The desired amount of gDNA template for PCR is ~0.1 ng/ul (final template concentration) so for a 30 ul PCR reaction use 3 ng of DNA.

Using Cells as Template: It is also possible to do colony PCR. The dilution of cells used in the reaction plays a critical role in the success of the amplification. Too many cells or components of certain media can inhibit PCR. For best results take a visible turbid overnight culture from a rich medium and dilute approximately 10,000-fold into the final PCR. The optimal dilution is somewhere between 1000 and 10,000 fold. Ideally, make a dilution series of the template cells. This PCR setup should include an initial denaturing step: 10 min at 94°C.

A typical PCR program is as follows:

After PCR, a few µl of the reaction needs to be run on an agarose gel to confirm the correct product amplified (around 1-kbp). A 1.5% or 2% gel works the best for this.

If the band is clean, then you can do a PCR cleanup spin column and submit the DNA for two different Sanger sequencing reactions (one with each of your PCR primers). If the band is not clean, you may be able to excise the band, gel purify, and use that sample for sequencing.

Analysis

You can use BLAST or a variety of other tools to identify your bacterium:

• Targeted Loci BLAST - select the appropriate database for your samples. Usually: rRNA/ITS databases > 16S ribosomal RNA sequences (Bacteria and Archaea)
  • BLAST result for Pseudomonas syringae:
    

Another option is EZBioCloud.

References

1. Baker, G.C., Smith, J.J. & Cowan, D.A. (2003) Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 55:541-55.
2. Burke, G.R., Normark, B. B., Favret, C., & Moran, N. A. (2009) Evolution and diversity of facultative symbionts from the aphid subfamily Lachninae. Appl. Env. Microbiol. *75*(16)5328–5335. https://doi.org/10.1128/AEM.00717-09