I am studying the diversity of organisms colonizing seagrass leaf surface and wish to target eukaryotic SSU, among other markers, using a metabarcoding approach.
I would recommend the primers described in Piredda et al. 2016 that target the V4 region of the 18S rRNA gene (Article Diversity and temporal patterns of planktonic protist assemb...
). We are using them now routinely with excellent results. Although the V9 region is also possible, I would rather recommend V4 which is longer and for which more sequences are available in the databases. See also :Article Comparison of coastal phytoplankton composition estimated fr...
We are currently running a project to propose a set of primers for different purposes.
what I can already tell you is that there is not THE or ONE primer pair that targets everything. Rather it depends on what you are interested in.
The region most people study now is the V4 region see the paper below.Article CBOL Protist Working Group: Barcoding Eukaryotic Richness be...
)and then there are plenty of options... universal primers or groups specific ones. universal ones are for example in
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.2009.04480.x/full
Article Coprophilic amoebae and flagellates, including Guttulinopsis...
but several others exist all with pros and cons...
in the end, if you have a good question and target the organisms of interest the primer pair shouldnt matter even if a lower diversity is recovered than with other primer sets...
Hope that helps!
More news on the proposed primers in a few months :)
Stefan
I would recommend the primers described in Piredda et al. 2016 that target the V4 region of the 18S rRNA gene (Article Diversity and temporal patterns of planktonic protist assemb...
). We are using them now routinely with excellent results. Although the V9 region is also possible, I would rather recommend V4 which is longer and for which more sequences are available in the databases. See also :Article Comparison of coastal phytoplankton composition estimated fr...
A colleague of mine has recently used 18S primers for sea grass leaf surface in the baltic sea. Might be worth checking out these primers.
"Eelgrass Leaf Surface Microbiomes Are Locally Variable and Highly Correlated with Epibiotic Eukaryotes". doi.org/10.3389/fmicb.2017.01312
You can use 18Sv4f and 18Sv7r from Meland & Willassen 2004
I used them to explore the diet of the carnivorous sponge Lycopodina hypogea .
They have quite a wide range of targets: From Copepoda, Mysida, Nematoda, Ophiuroidea to Polychaeta.
Best,
Article Preliminary DNA-based diet assessment of a gutless carnivore...
I strongly agree with Stefan that there is no "ONE primer pair that targets everything".
What sequencing platform will you be using, because the length of primers are also important to consider, if you want to use overlapping for MiSeq for example? A colleague of mine had very good results with these primers targeting marine benthos, while avoiding some planktonic taxa, targeting V2: Article Worldwide Analysis of Sedimentary DNA Reveals Major Gaps in ...
For longer amplicons sequenced with 454, we recently used the primers F566 and R1200 with very good results in terms of universality, but they are not as suitable for MiSeq since the amplicons are too long to overlap: Article Characterization of the 18S rRNA Gene for Designing Universa...
If you're using illumina MiSeq sequencing, keep amplicons short. Leray and Knowlton's COI is a good length (2015 paper: http://www.pnas.org/content/112/7/2076).Stoeck et al. 2010 use 18S v4 region which is about 300 bp. However, choose the right oligotags- as these can make amplicons a bit too large for decent overlap. I've used both amplicons and have got a very broad range of eukaryotes from the marine environment.
Hope it works well!
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