Hi Tatiana,

a lot of studies proposed fungal primer pairs to NGS approaches (Ihrmark et al., 2012; Toju et al., 2012; Blaalid et al., 2013). Our latest results confirmed that the primers ITS1F / ITS2 (Gardes & Bruns, 1993) are the most convincing, using mock fungal communities. The Toju’s primers seem also valid. The primers developed by Ihrmark are good also (better covering for Ascomycetes), but they also amplify the plants (eg oak). Pay attention to the environmental matrix you use ...

Be careful, if you include your bacterial 16S amplicons in the same run as your fungal ITS amplicons, because Miseq technology requires that your amplification products are fairly similar sizes ...

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for Basidiomycetes: application to the identification of mycorrhizae and rusts. Mol Ecol 2: 113–118.

Toju, H., Tanabe, A. S., Yamamoto, S., & Sato, H. (2012). High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples.
Ihrmark, K., Bödeker, I. T., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., ... & Lindahl, B. D. (2012). New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communities. FEMS microbiology ecology, 82(3), 666-677.

Hi : for fungi the best primers for identify fungi sequencing ITS1 and ITS4.

If I remember correctly ITS1 and 5.8S are a common pairing for MiSeq and the ones used by JGI.  A major consideration is the length of the read as you will want sufficient coverage to be able to confidently align the forward and reverse reads; probably nothing over 350-400bp. Depending on the number of samples you have you may be limited to primers the sequencing company already has, but most will develop new primer sets for a nominal fee if you have a lot of samples (normally a flat rate of about $200). In any case you should get hits from all cultured species anyway since they will have all ITS sequences, you'd just be missing clones that only covered specific regions, but that occurs regardless of the region you choose. Once you have data check out https://unite.ut.ee/repository.php which is a great database for fungal taxonomy assignment that is updated regularly and has formats for pretty much all pipelines. In all reality how you handle the data after sequencing is more important and any reviewer who puts up a big fuss about primers (especially commonly used ones) is probably just having a bad day. 

Hi,

Actually it depends on the community you have, because each primer pair has an efficiency which is different on various fungi. 

You should take a look at Tedersoo et al work (recent), whichwill give you good clues.

(see attached links)

http://science.sciencemag.org/content/349/6251/936.4.short

http://link.springer.com/article/10.1186/s13742-015-0074-5

Hi everybody

Thanks a lot.

I can't use two pairs because this method isn't cheap. Besides, I have too many samples and also the analysis of bacterial diversity. On the other hand, my study includes other topics and the budget is limited however, I know that it would be the best option if my study only was about diversity.

Hi Tatiana,

a lot of studies proposed fungal primer pairs to NGS approaches (Ihrmark et al., 2012; Toju et al., 2012; Blaalid et al., 2013). Our latest results confirmed that the primers ITS1F / ITS2 (Gardes & Bruns, 1993) are the most convincing, using mock fungal communities. The Toju’s primers seem also valid. The primers developed by Ihrmark are good also (better covering for Ascomycetes), but they also amplify the plants (eg oak). Pay attention to the environmental matrix you use ...

Be careful, if you include your bacterial 16S amplicons in the same run as your fungal ITS amplicons, because Miseq technology requires that your amplification products are fairly similar sizes ...

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for Basidiomycetes: application to the identification of mycorrhizae and rusts. Mol Ecol 2: 113–118.

Toju, H., Tanabe, A. S., Yamamoto, S., & Sato, H. (2012). High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples.
Ihrmark, K., Bödeker, I. T., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., ... & Lindahl, B. D. (2012). New primers to amplify the fungal ITS2 region–evaluation by 454-sequencing of artificial and natural communities. FEMS microbiology ecology, 82(3), 666-677.

Hi Tatiana

The primer ITS is a universal primers, and Forward ITS1 - reverse ITS4

Hi Tatiana

I am working on soil samples and the primers that I used was ITS5 and 5.8S-fungal, but this pair for Basidiomycota fungi. So, it depends on what exactly you want. Then you can decide. 

Thank you everybody.

Definitely i used ITS3-ITS4 and I obtained a good coverage.

Thank you very much!

Hi please take a look this article for your consideration, http://www.tandfonline.com/doi/abs/10.3852/11-027?journalCode=umyc20

Thank you!

and what about ITS7 AND ITS 4 it can be used for miseq or not