Hi,
I recently decided to opt for the ITS3_KYO2/ITS4 primer combination in the Toju et al., (2012) High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples. PLos One. In the paper it specifies and tests a mock community composed of Ascomycetes and Basidiomycetes, but I wonder if anyone has used these primer sets and got a wider coverage of the fungal community from environmental samples?
The paper by Balint et al., (2014) An Illumina metabarcoding pipeline for fungi. Ecology and Evolution that used ITS3_KYO2/ITS4_KYO3 says that they lost a number of reads during the clean up and only managed to assign 33% of OTUs to fungi.
I also then found a paper by Asemaninejad et al., (2016) New Primers for Discovering Fungal Diversity Using Nuclear Large Ribosomal DNA. PLos ONE that compared a new primer set with the Toju et al (2012) primer set. Their new primer set target the LSU and while they performed better than the Toju primer set, is it better to target the ITS region where a lot of the research has previously been performed so that we have a larger database to assign taxonomy?
I think the answer depends on what environmental samples you're working it. If you work with plant-related environments, it's best to avoid the ITS2 region since primers designed for that region tend to amplify lots of plant DNA.
Thank you for your comment I really appreciate it. From what I have read around the subject, the primer set used by Toju et all (2012) (ITS3_KYO2/ITS4) appeared to
Perform well at producing minimal plant amplification.
I was moving away from the ITS1 region as I read that due to a large intron at the end of the SSU for some fungal groups ( ie Ascomycota) this can generate applicons of varied lengths possibly too long for illumina MiSeq NGS (2x300bp paired end reads) and possibly bias the amplification of certain fungal groups.
There is still a lot of debate around this issue. A lot of the papers I've seen coming out of Europe advocates for ITS2, but it's mostly theoretical that it works better. North American groups tend to stick with ITS1 that still performed well. In the end, as long as you get good amplification and good data to work with then you should work for what's easiest.
I'm not a fan of the ITS3_KYO2/ITS4 because it was designed for Ascomycota and Basidiomycota. Why such a limit if you were going to study total fungal communities?
Hello,
I agree with your last point about the coverage issue of this primer set when wanting to target the broader fungal community. I guess it brings us back to my original question of whether anyone has used this set on environmental samples and got a wider coverage of the fungal community other than Ascomycota and Badisomycota?
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