non-fungal sequences in ITS2 analyses

[Nicholas_Bokulich]

Hi @carydavies ,

Indeed, in a typical ITS analysis of fungal communities (as in the tutorial that you mentioned), non-fungal features should be filtered out. Because if you are looking at the mycobiome, plants, animals etc are non-target and not part of the microbial community.

But in your case you are not only looking at the mycobiome, you are doing a diet metabarcoding analysis as well.

So for your case as you want two analyses for the price of one I recommend: (1) filtering out non-fungal non-microbial features for mycobiome analysis; (2) in a separate analysis filtering out all microbial fungal features for diet analysis (keeping mushrooms, which would be dietary).

@carydavies

If ASVs assigned to non-fungi are true ITS2 sequences and not just off target amplifications, is it incorrect to include them in downstream analyses?

Yes, because these non-fungal ASVs are separate from the microbial community that you are trying to describe; they are not living, interacting members of the gut mycobiome but instead are dietary constituents. And similarly, intestinal fungi should be removed from the dataset for diet metabarcoding analysis because these are (presumably) not intentional dietary constituents so if you are trying to describe what an animal is eating these ASVs should be removed. It is about cleanly separating these constituents to describe different compartments (dietary intake vs. gut mycobiome community).

@carydavies

If these non-fungal taxa are removed, as they often are, could this bias the relative abundances of fungal taxa?

If they are not removed it will bias the relative abundances of fungal taxa, as relative abundance could be highly skewed by differences in diet. If they are removed, it should not create a bias as long as sufficient fungal reads remain and you normalize appropriately afterwards.

@carydavies

database which is more populated than UNITE for non-fungal taxa (for example using blast with NCBI-nr)

I have not used it yet so cannot share my experience with it, but Eukaryome seems to fit the bill. They even compile QIIME 2-compatible files:
https://eukaryome.org/qiime2/

and other forums users have kindly provided pre-trained naive Bayes classifiers of this database:

Pretrained EUKARYOME(v1.9) Database for Eukaryotic Taxonomy in QIIME2

Hello, QIIME2 users. I have recently trained the newly released EUKARYOME database in QIIME2 format. More information can be found on the official website (https://eukaryome.org/). The EUKARYOME database integrates other databases alongside UNITE version 9.0. Based on my experience, it has been more effective in detecting unassigned species that UNITE ver10.0 couldn't assign. Of course, each has its pros and cons, but I hope you find it useful for your research. I am sharing the classifier fi…

@carydavies

Since these sequences were amplified with primers developed for fungi, they would not represent a comprehensive set of non-fungal taxa, but could they still be recognized as a subset of eukaryotes other than fungi present in the samples?

I recommend checking this. If you can acquire a reliable reference set of non-fungal ITS sequences (e.g., eukaryome, and filter out the fungi) you could use a primer evaluation tool to see how much coverage etc your primers have of different groups.

Good luck!