Previously, I expressed some concern about size variation in my environmental fungal ITS PCR libraries. I’m still concerned about that, but I have an additional concern. The ITS region can’t be aligned, and I’m partial to phylogenetic approaches to pretty much everything. So maybe ITS is not for me?

So, I asked Twitter again…

[View the story “What the fungi do I do with my ITS library? (Pt. 2)” on Storify]

In summary, I don’t think that I can use ITS given the size variation that I see, and I’m not sure that I want to, given the fact that you cannot align it to do phylogeny-based analyses.

28S (or LSU) is a reasonable alternative to ITS that has two big downsides: 1) the reference database is much smaller than the ITS reference database and 2) it does not provide the fine-scale taxonomic resolution that ITS does.

Rachel Adams referred me to Amend et al,  in which they use both. I’ll have to look into this approach…