16S Probes - Frequently Asked Questions

1. Where is the 16 Probes database described?

   ES Wright et al. (2014) "Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification." Applied and Environmental Microbiology, doi:10.1128/AEM.01685-14.

2. What genera are available in the 16S Oligos tool?

   All 1,943 archaeal and bacterial named genera in the RDP II database (10.30) have probes available.

3. Have you tested all of these probes?

   No, these probes were designed automatically using a state of the art model of probe hybridization. As with any newly designed probe, it is recommended to experimentally verify that they work as expected using flow cytometry (or an equivalent method) with the target organism.

4. What is the difference between the single probe and dual probes options?

   Two FISH probe options are available for each genus:

   • Single 16S FISH Probe:

     This is the traditional approach of using a single probe to target the group of interest. In many cases DECIPHER predicts that these probes will not be sufficient to adequately identify the target genus using a single probe without potential false positives.

   • Dual 16S FISH Probes:

     In this case a set of two probes are designed to have minimal cross-hybridization overlap with non-target genera. Here the probes are labeled with different fluorophores and the overlap of both colors is necessary for positive identification. For example, if one probe was labeled with Cy3 (red) and the other with fluorescein (green), then the overlap along with DAPI (blue) would appear white.

5. How do I interpret the output page?

   After selecting a genus the output page displays the top 10 probes with highest specificity to that genus. The probes are listed along with their starting E. coli positions, and respective coverage of the target group. The predicted melt point of each probe is listed, which can be used as guidance for a formamide concentration to be used during hybridization. Possible cross-hybridizations are shown in order of their potential risk for causing a false positive identification. In the case of dual probes information is only listed for the probe (one or two) that has the least potential to cross-hybrize with each non-target, as this probe is the limiting factor experimentally.

6. Why are there competitor-oligonucleotide probes shown?

   If there are any mismatched non-targets with a HIGH risk of cross-hybridizing then potential competitor-oligonucleotides are provided for further consideration. The addition of these unlabeled probes may block hybridization with one or more non-targets. For dual probes, competitors are given for the probe with the lowest risk of cross-hybridization, since this will probably be the easiest probe to block. Competitive probes should be assessed based on the expected risk level of the non-target, and their potential to block hybridization to only the non-target. A link is provided to mathFISH, which offers an online tool for confirming the adequacy of competitive probes.

7. How should I use these probes in an experiment?

   Probe sequences can be ordered from many online companies, including Sigma-Aldrich and Integrated DNA Technologies (IDT). We used the probes at 250nM final concentration, with a hybridization of 18 hours at 46°C in a buffer of 35% formamide (v/v) and 1M [NaCl]. Non-targets listed as HIGH risk can often be eliminated using a competitor probe. Non-targets shown as MODERATE or LOW risk will probably not cross-hybridize if stringent enough hybridization conditions are used.

8. How can I verify the specificity of the probe(s) in silico?

   All non-target genera predicted to have at least a modest chance of causing a false positive are listed with mismatches highlighted. Links to probeCheck and the RDP's ProbeMatch are provided that will automatically search their sequence repositories using the probe(s) listed. These tools enable independent confirmation of probes' in silico specificity to the target group.

9. How can I predict the melt curve of the probe(s) in silico?

   A link is provided to mathFISH, which provides multiple online tools for analyzing FISH probes in silico. Alternatively, ProbeMelt can be used to predict the melt curve.