Suggestions for ASV table with ITS (fungi)

Hi everyone: I am wondering the following: I am curious how many ways people do things, as there are multiple ways.

  1. How do you with QIIME2 deal with ITS (fungi) to make sure the ASV (amplicon sequence variant) taxonomy is assigned properly?

  2. Any tutorials you can recommend for more recent analyses? I noticed some older ones.

  3. Which version of UNITE to you recommend using? See two here:

  4. Did you trim before running DADA2 and if yes why and how? (assuming everything but primers removed, demultiplexed, and barcodes removed).

Best wishes,
Laura

Hi @Laura,
Hopefully multiple users will speak up on this topic so you can learn from as many perspectives as possible, because this statement is very true:

Sure enough. I will try to describe a few ways:

Taxonomy classificaiton is pretty much the same as for 16S (it’s the upstream steps that differ)… q2-feature-classifier has multiple different classification methods and they all work quite well for fungal ITS… see this paper for details (adjusting defaults can improve precision for fungal classification):

I wrote this tutorial a couple years ago and I am not sure much has changed, so I still recommend this basic workflow, but let’s see if others propose some upgrades:

I recommend the latest version… whether to do fungi only or all euks depends on how many non-fungal reads you expect. In general, at least a few plant (or other non-fungal) ITS reference sequences is enough to provide an outgroup for identification of non-target hits.

We actually benchmarked a few different versions of UNITE recently, take a look here for details:

YES, definitely! This is a big issue with ITS… the hypervariable length means that read-through can be a common issue (depending on your read length), so you need to trim the forward primers AND sometimes the reverse primers and adapters that appear in the reads (for more details, this has been discussed in other topics on this forum, maybe also the tutorial linked above).

There are two main ways to do this in QIIME 2:

  1. q2-cutadapt can be used to trim at primer sites (forward and reverse)
  2. q2-itsxpress will trim at the ITS site (discarding the conserved rRNA gene regions that flank the ITS). This plugin has been discussed quite a lot on this forum if you run into difficulty or want to read more about it. See also this tutorial: Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences

I hope that helps!

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