# fungal ITS analysis with QIIME2: current recommended workflow?

**URL:** https://forum.qiime2.org/t/fungal-its-analysis-with-qiime2-current-recommended-workflow/30809
**Category:** General Discussion
**Tags:** its, vsearch
**Created:** [July 12, 2024, 9:10am UTC](https://forum.qiime2.org/t/fungal-its-analysis-with-qiime2-current-recommended-workflow/30809 "2024-07-12T09:10:55Z")
**Posts on this page:** 1
**Showing post:** 2

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### Author: ![salias](https://forum.qiime2.org/user_avatar/forum.qiime2.org/salias/32/18594_2.png) [@salias](https://forum.qiime2.org/u/salias)
#### Post date: [July 12, 2024, 10:42am UTC](https://forum.qiime2.org/t/fungal-its-analysis-with-qiime2-current-recommended-workflow/30809/2 "2024-07-12T10:42:04Z")

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Hello @Rob_DNA ,

There is a lot to unroll here. I'm not an expert, but my first steps with metataxonomy are precisely with ITS sequences. So I will share my thoughts on the points on which I feel "confident" enough to discuss them.

> [@Rob\_DNA](#):
>
> ITS extraction before denoising appears to work well, as I obtain a higher proportion of filtered/merged reads from `dada2`. Agreed that this makes sense?

Agreed. Since ITS sequences vary notably in length (they are indel-rich regions) I use ITSxpress for two purposes:

1. The original purpose described in the tutorial:

> [@Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences](https://forum.qiime2.org/t/q2-itsxpress-a-tutorial-on-a-qiime-2-plugin-to-trim-its-sequences/5780/1):
>
> Previous work by [Nilsson et al. (2009)](https://doi.org/10.1111/j.1574-6968.2009.01618.x) showed that removing the conserved regions around the ITS results in more accurate taxonomic classification.

1. My other purpose: dynamic quality filtering. I don't want to truncate sequences to a fixed length because of the nature of ITS sequences. So I take advantage of the fact that ITSxpress is trimming sequence sections not belonging to ITS, which turn out to be sections with low quality. Consequently, DADA2 works much better without me having to specify further truncation or trimming parameters. I don't know if this is the best way to do things, but at least it's the way that works best for me.

> [@Rob\_DNA](#):
>
> However, not everyone agrees with this, see e.g. [ASV vs OTU for fungal ITS](https://forum.qiime2.org/t/asv-vs-otu-for-fungal-its/30735/2)

I am an advocate of the idea that for ITS sequences we should use ASVs without further clustering. On this note, I really like [this Colin answer](https://forum.qiime2.org/t/asv-vs-otu-for-fungal-its/30735/4) in the post you cite.

> [@Rob\_DNA](#):
>
> However, there are several UNITE releases (97%,99%, dynamic clustering) and I'm completely sure which to use..

Although I also use Colin's pre-trained classifiers, I'm afraid I cannot help here since I assign taxonomy to unclustered ASVs. However, just in case you want to know, I'm currently using 99%, all eukaryotes, version without "s"¹ database.

--

¹ Even after reading release descriptions in the [UNITE webpage](https://unite.ut.ee/repository.php), I'm not sure about what the difference is between the versions with and without "s".

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