I'm getting ready to place an order for barcoded primers that will be used for Illumina sequencing of host-associated and non-host-associated fungal communities. After doing some research on primers, I'm leaning toward ordering the ITS1f / ITS2 primers, which target the ITS1 region. (ITS1f: CTTGGTCATTTAGAGGAAGTAA; ITS2: GCTGCGTTCTTCATCGATGC; references for both are below.) These are the "Earth Microbiome Project ITS primers", described here and in Walters et al (2016), which is helpful because there will be a lot of pre-existing samples that were sequenced with these primers that we could compare against.
Before ordering I wanted to check if anyone had more recent recommendations on a good fungal ITS primer choice? It's been a few years since I've done any fungal community analysis work. Since these will be used to target host-associated and non-host-associated samples across a wide array of environment types, broad coverage is important. It's also important that there is existing taxonomic reference data (UNITE should work well for ITS1f / ITS2), and total amplicon length is important as we'll be starting with 2x300 Illumina runs.
The ITS1f / ITS4 pair also sounds nice since it covers both the ITS1 and ITS2 regions, but the amplicon is too long to be generally useful with Illumina sequencing (e.g., this came up on the forum here).
Another question: has anyone had experience (good or bad) with barcoding the ITS1f primer instead of the ITS2 primer? The EMP protocol puts the barcode on the ITS2 primer, but it seems like putting the barcode on the ITS1f could leave the door open to using this with the ITS4 primer in the future, e.g., if Illumina reads get considerably longer. (We're hoping to have this pool of barcoded primers for a while.)
ITS1f primer: Gardes M and Bruns T. 1993. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113–118.)
ITS2 primer: White T, Burns T, Lee S, and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p 315–322. In Innis MA, Gelfand DH, Sninsky JJ, and White TJ (ed), PCR protocols: a guide to methods and applications. Academic Press, San Diego, CA.
For reference, this diagram shows what the different primers target. I sourced this from one of @Nicholas_Bokulich's posts, and the original source is here.