Just a few days ago we discussed difficulties encountered when investigating host-associated mycobiomes by a metabarcoding approach suggesting some solutions.
Primers need to be generic enough to amplify target DNA from most taxa (including those still unknown), but also specific enough to avoid the amplification of DNA from non-target host (e.g., human or plant DNA).
Well, sticking to a subject, valuable experimental data has been freshly published by Scibetta et al.. The authors tested five ITS1 and ITS2 primer sets possessing the most desirable features in terms of fungal coverage and specificity. These primer sets were evaluated by analyzing the fungal microbiota associated with leaves of four globally ubiquitous plant crops (Vitis vinifera, Triticum aestivum, Olea europaea and Citrus sinensis).
The specificity of the selected primers was extraordinary, a percentage of fungal reads ranged from almost all to 65% of the total sequences. Primer sets targeting ITS1 region were consistently more specific compared with those targeting ITS2 region.
Even though all primer sets detected a similar number of taxa, a single set captured only a fraction (~50%) of the actual genetic diversity. The coverage was increased to 70-80% by combining results from two different primer sets, especially when ITS1 primers were combined with primers targeting the ITS2 region. It is worth mentioning that several fungal taxa were preferentially or exclusively detected by some primer sets, generally recurrent on more investigated hosts.
Taken together, it is obvious that a perfect primer set to investigate the whole fungal diversity does not exist and that most of the currently available metabarcoding studies have only revealed a fraction of the fungal diversity.
Nevertheless, this highly interesting reading provides information of the specific features of different primers important for the selection of the most suitable according to the host, the environment and the objective of the analysis.