Hello, we have a QIIME 2 pipeline applied to 16S data developed by an alumni student of our master. The first step examines the quality of metabarcoding sequences using the function demux summarize. The second step infers Amplicon Sequence Variants (ASVs) using the function qiime dada2 denoise-paired. The third step classifies reads by taxon using a pre-fitted sklearn-based taxonomy classifier from (SILVA). Additionally, there is an extra step that generates interactive alpha rarefaction curves by computing rarefactions between min_coverage and max_coverage.
Now, I am looking to adapt this pipeline for ITS (fungi). I believe the first step using demux summarize can remain unchanged and I think also there is no need to touch the extra step that generates interactive alpha rarefaction curves!! . However, I am uncertain about the necessary modifications for the subsequent steps (DADA2 and taxonomy). Could you please guide me on the specific changes required in these steps to apply the pipeline to ITS data?
I hope you can guide me because I'm a little lost, as I'm a beginner in this field.
NB : I already saw this tutorial, but it's still not entirely clear to me what the main changes are compared to16s : Fungal ITS analysis tutorial
@colinvwood , thank you for responding
Do you mean I only need to use UNITE in taxonomy step ( third step) ? I don't need to modify any other step in the workflow ?
if it's the case where I can find the pre-trained classifier for UNITE trained for use with qiime2-2022.8.3 please ( the .qza file). I found this link but I don't know which one I should download : UNITE - Resources
I have another stupid question if you don't mind : For instance I work with qiime2-2022.8.3 , if I want to work with the latest version of qiime2 I should modify the scirpt forming my pipeline ? or I just have to update the conda env and then run the previous script without any modifications
You don't necessarily need to use the UNITE database that the tutorial recommends but you do need to use some classifier that is made for fungal ITS sequences.
While the workflow will be the same it won't be as simple as just running some script that was used for 16S sequences--different barcodes might be needed during demultiplexing, different primers trimmed, different dada2 parameters, etc. If these things are new to you then check out the moving pictures tutorial.
If you want to get a newer version of qiime2, yes you will need to make a new conda environment. Those instructions are here.
@colinvwood, thank you very much for all these clarifications. However, since I'm a beginner, I want to use the same classifier as the one in the tutorial. Could you please guide me on where to find the pre-trained classifier for UNITE, specifically trained for use with qiime2-2022.8.3? I'm looking for the corresponding .qza file on this website (UNITE - Resources). Could you please advise on which one I should download? Thank you!