Hi Jorge,

ITS1F/ITS2 are mostyl general primers. You will get a lot of ectomycorrhiza as well. However, by NGS you will not get absolute quanty, only relative. For absolute I would reccommend qPCR. If you really want specific primers and you are interested in quantity try to look at this parer of my coleague:

Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA

Karol Krak, Martina Janoušková, Petra Caklová, Miroslav Vosátka, Helena Štorchová

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 3630-3637 2012

It is difficult to provide an answer without more knowledge about the experiment.

Target genes for fungal diversity are usually

- SSU rRNA genes.

- ITS sequences.

Choice of primers should take into account coverage and specificity

coverage : every fungal clade amplified

specificity : only fungal clades amplified

Now specificity is important if you expect lots of NOT fungi in your samples. Otherwise, because NGS provides millions of reads, a 50% not fungal reads is not a problem, they can be easily filtered out.

Coverage : well, I am presently working on this problem :-)

I expect an answer within a week.

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SSU versus ITS

SSU genes have a very good phylogenetic signal, but a poor taxonomic resolution at the species level for many genera of fungi.

ITS has a much better taxonomic resolution, but almost no phylogenetic signal. As a result an ITS sequence with no similar sequence in you reference database ends up as "unknown" and no idea of the genus, family, order...

As a matter of fact if you want taxonomic precision, go for the IGS. ITS have a selective pressure (2D structure required for pre-rRNA maturation) which is absent from IGS. But the IGS database is virtually None.

In conclusion much depends if the question is identifying each read at the species/genus level or if what you want are comparing samples for taxonomic diversity at the species level, even if in the end you cannot put a taxonomy on many reads.

If you should go for ITS

- F primer in SSU, R primer in 5.8S, Illumina should be fine.

- F primer in SSU, R primer in 28S, Illumina paired-end should be fine to get its1 & its2, otherwise Roche 454 (may be BioPac, but I have no experience).

Hi Richard

Many thanks for your great explanation. Our objective is to characterize diversity of arbuscular mycorrhizae and pathogens associated to roots of Vitis vinifesa in Chile. We want to use the methodology proposed by Yu et al. 2012 (http://link.springer.com/article/10.1007%2Fs11104-012-1188-5?LI=true). They propose to use primers ITS1F and ITS2 and your response confirms to me that is the best choice.

Some food for thought:

http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6941.2012.01437.x/pdf

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0040863