Hello,
I’m sorry if this is a silly question, but I have been looking for information about those primers and I have found contradictory data, or maybe there is something I am not understanding very well.
The question is: Is the pair of primers 515f (GTGCCAGCMGCCGCGGTAA) 806r(GGACTACHVGGGTWTCTAAT) suitable both for bacteria and archaea?
You will get Archaea but not all. If you want Archaea, you’re better off using primers designed for them
Hello.
Thanks for your answer. We want both Bacteria and Archaea, I guess this complicates things.
What about the primers proposed by Earth Microbiome Project? (515FB-806RB, FWD:GTGYCAGCMGCCGCGGTAA; REV:GGACTACNVGGGTWTCTAAT )
http://www.earthmicrobiome.org/protocols-and-standards/16s/
It would be for a faecal study.
I’m confused because in some papers they claim that 806R (GGACTACHVGGGTWTCTAAT) is only for bacteria, but in EMP protocol they say that the degeneracy added to improve detection of Archaea is on 515F, not 806r. I’ve made some test with silva’s TestPrime and this seems to be confirmed, but I don’t understand what happens with those papers then.
in silico tests are a decent way to figure out if they will hit your expected organisms. If I recall correctly it’s the thaumarchaeota that the primers don’t hit well (not sure if that has been fixed with the EMP alteration or not). When you’re looking at these, also keep in mind that you need the TM to be above 65C if you are using target primers as sequencing primers (the strategy that both Caporaso and Kozich use). So, even if the degeneracies mean the Thaumarchaeota amplify, if the primers that match them are too cool they won’t sequence.