Fungal ITSx and qiime2

Hi

We are not microbial ecologists, but learning :slight_smile:
We are analyzing fungal amplicons from soil samples, and are wondering whether or not to extract ITSx from the amplicons, and if so where in the pipeline to do so.

We have single-end Illumina reads, and have run the following:

  • qiime demux emp-single
  • qiime dada2 denoise-single

Does it make sense as a next step to extract ITSx with the repset from dada2, and then go on to align (mafft), mask, and then construct phylogeny using raxml ?

If yes, how do we read the ITSx fasta sq back into a qza file?

Apologies for naive questions

Charles

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Welcome! You came to the right place.

This is not essential, but can help depending on the primers you used. Some primers leave very little of the flanking rRNA gene regions in the amplicon — others do, and this can impact taxonomy classification results. I have done ITS analysis without ITSx in the past without issues. (the big issue that can occur, though, is when your reads are longer than the amplicon, as described in this forum post, which is very relevant to your questions).

Incidentally, I just discovered (via this post) a new 3rd party qiime2 plugin, q2-itsxpress, that will perform ITS trimming for you! So no need to export, trim, then re-import.

Check out q2-itsxpress… it looks like this happens after demultiplexing but before denoising. @Adam_Rivers may be able to tell us more!

ITS alignments are not phylogenetically informative except possibly between very closely related species — so don’t do the alignment. If you are just doing the alignment for diversity estimates (e.g., with UniFrac), just use non-phylogenetic methods. You could also check out q2-ghost-tree (and @Jennifer_Fouquier can give more details — I’m not sure whether that plugin is still in development mode)

Don’t apologize, that’s what we’re here for. Thank you for posing very clear questions. :smile:

I hope that helps! :mushroom:

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We are planning to make an announcement about q2_itsxpress tomorrow or maybe Monday once the most up to date version is on bioconda. I’ll let you know when its up.

Adam

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Hi @Charles_Hauser and @Nicholas_Bokulich!

Great reply. I agree that non-phylogenetic methods are better than doing the alignment for the reasons Nicholas mentioned. You can use the ghost-tree tool on its own, or I can help you get the appropriate tree (feel free to email me at [email protected]… fungal research is so small I really don’t mind helping while I’m behind on development! :grin:). I have not had the time to finish the q2-ghost-tree plugin yet, but need to do that ASAP. These nudges help!

For a super fast summary of ghost-tree, see this tweet or check out the paper.

Good luck! -Jennifer

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Thanks for all the suggestions !
I’ll follow up on q2-itsx, and q2-ghost-tree

Charles

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Hi Charles,

Here are the instructions for installing Q2-itsxpress Assuming that you installed Qiime2 in a conda environment.

Installation
Activate the qiime environment
source activate qiime2-2018.6

Install the base package ITSxpress v1.6.1 from bioconda
conda install -c bioconda itsxpress

Install the Qiime plugin Q2-itsxpress v1.6.1 from pip
pip install q2-itsxpress

Activate the module in Qiime
qiime dev refresh-cache

Use

you can now use the commands for single-ended or paired end data:
qiime itsxpress trim-single
qiime itsxpress trim-paired

For more details see the documentation here:

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I hit post too soon. The Bioconda build is still pending. The instructions should work once my pull request is merged by the Bioconda maintainers.

If you need to run it sooner you can install the dependencies with Conda and ITSxpress with PIP:
conda install -c bioconda hmmer vsearch bbmap biopython
pip install itsxpress
pip install q2-itsxpress

Update
ITSxpress v1.6.1 is now available on Bioconda.

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An off-topic reply has been split into a new topic: Q2-itsxpress reformat.sh error

Please keep replies on-topic in the future.

Adam

Thanks for quick response - program is running fine now.

Charles

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