Bacterial 16s rRNA gene is the most widely used molecular marker to study the diversity of microbial communities. However, multiple primer sets have being used in different combinations to target variable regions from V1 to V9. What primer set is good for second-generation DNA sequencing (e.g, amplicon sequencing by 454 pyrosequecncing )?
For human microbiome study, the primer set 27F/R338 (V1-V2) was recommended, while for environmental samples, 515F/R806 (V4) was recommended (Kuczynski, et al,2012, Experimental and analytical tools for studying the human microbiome, Nature Review Genetics). Short-read length is not a problem because it is found that short sequences have sufficient resolution for the accurate taxonomic assignments as the full-length sequences do (Liu,et al.,2007. Short pyrosequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res 35: e120).
Here is an original paper about using 515F/806R. This primer set amplifies both bacterial and archaeal sequences. (Caporaso, 2011, Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample, PNAS)
Too many papers have being talking about this topic while a not an conclusion achieved. Many people prefer the primers targeting V3 or V6 region. For 454 sequencing, the relative long read length (400-500bp) is a big advantage, which makes it possible to use primer pairs targeting more than one region. Some good examples include those using V1-V2, V1-V3, V3-V4, V5-V6.
http://www.biomedcentral.com/1471-2180/10/206
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0002836
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932916/
No definite answer, everyone you´ll ask will give you a different region(s). Take a look at this paper: Huse SM, Dethlefsen L, Huber JA, Welch DM, Relman DA, et al. (2008) Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing. PLoS Genet 4(11): e1000255. doi:10.1371/journal.pgen.1000255
For human microbiome study, the primer set 27F/R338 (V1-V2) was recommended, while for environmental samples, 515F/R806 (V4) was recommended (Kuczynski, et al,2012, Experimental and analytical tools for studying the human microbiome, Nature Review Genetics). Short-read length is not a problem because it is found that short sequences have sufficient resolution for the accurate taxonomic assignments as the full-length sequences do (Liu,et al.,2007. Short pyrosequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res 35: e120).
Here is an original paper about using 515F/806R. This primer set amplifies both bacterial and archaeal sequences. (Caporaso, 2011, Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample, PNAS)
Hi Daniel, i'm having a similar problem working this one out, and I agree that there is no definite answer. Trying to choose between v3-v4 and v3-v5 as although v3-v5 may give a longer read length it still does not necessarily mean clearer results and reads....
I have also read that the 515F/806R primers for V4 region is a good region to target for soil and sediment analysis. My question is should more than more variable region be targeted and would bacterial biofilms influence the target region?
- Badges
- Science method