Suggestions for ASV table with ITS (fungi)

[Nicholas_Bokulich]

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:

@Laura

I am curious how many ways people do things, as there are multiple ways.

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

@Laura

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

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):

Optimizing taxonomic classification of marker-gene amplicon sequences with...

@Laura

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

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:

Fungal ITS analysis tutorial

Fungal ITS analysis, mock communities, and more fun NOTE: This tutorial was written in a QIIME 2 2018.11 environment. It is not guaranteed to work with earlier or later versions of QIIME 2. This tutorial was compiled as a working exercise for a QIIME 2 workshop in December 2018, and does not represent the only possible fungal ITS workflow with QIIME 2, or even a benchmarked protocol recommendation. See other tutorials, e.g., the q2-itsxpress tutorial for other fungal ITS analysis options in QII…

@Laura

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

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:

RESCRIPt: Reproducible sequence taxonomy reference database management for...

@Laura

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

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!