I am planning analysis on a complex microbial community containing high number of species of bacteria and archaea using 16 rDNA sequencing. On previous experiments I have used two different couples of primers to identify them independently, but the use of the same primers could allow a direct comparison of the relative abundance of these two groups of organisms. Are there papers in literature reporting the use of the same primers to check both bacteria and archea?
Thank you.
In Klindworth et al. (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. Jan 2013; 41: e1. doi: 10.1093/nar/gks808
you can find a large list of in silico evaluated universal, specific bacterial and specific archeal primers for amplifing both short and long 16S rRNA fragments (so suitable for 454, illumina and full lenght clones).
Cheers
This article in PLoS ONE reports some primer pairs with good coverage for both bacteria and archaea:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007401
More and more studies are using V4 primer set because it targets both bacteria and Archea, and is short (~250 bp) (good for Illumina to get millions of reads) but also long enough to get accurate taxonomic resolution. This one has been used by Earth Microbiome Project. http://www.ncbi.nlm.nih.gov/pubmed/20534432
Below is a review paper talking about the use of 16S primers. Although there is no primers without bias, V2 primer is found good for human microbiome and V4 is good for environmental samples.
http://www.nature.com/nrg/journal/v13/n1/full/nrg3129.html
Hi Stefano,
This would depend on what your downstream application for amplified rDNA is. If you do not need the full length gene, then I suggest universal primers 787F and 1391R for amplifying the V5 to V8 region of 16S. This will give you the ability to classify the organisms all the way down to the genus and sometimes species level. Here is a cool paper where they compare relative abundance of archaea and bacteria with these primers:
http://www.pnas.org/content/early/2012/09/26/1207574109.abstract
NB: since V5-V8 is ~600bp long, you will need to use a sequencing method that can produce such a long read. I just used this primer set for 454-pyrosequencing and got very nice results for archaea and bacteria.
I did not know a common primer coving both archaea and bacteria, may be such primer set is not exist. from my perspective, the best way to do simultaneous quantification of bacteria and archaea quantification is to do short-gun whole genome sequencing instead of 16S amplicon sequencing.
Still I think this paper will add value to such quantification with 16S amplicaion. It describe the method to combine different primers together for NGS sequencing.
Kittelmann, Sandra, Henning Seedorf, William A. Walters, Jose C. Clemente, Rob Knight, Jeffrey I. Gordon和Peter H. Janssen. “Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities”. PLoS ONE 8, 期 2 (2013年2月8日): e47879. doi:10.1371/journal.pone.0047879.
In Klindworth et al. (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. Jan 2013; 41: e1. doi: 10.1093/nar/gks808
you can find a large list of in silico evaluated universal, specific bacterial and specific archeal primers for amplifing both short and long 16S rRNA fragments (so suitable for 454, illumina and full lenght clones).
Cheers
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