We are not microbial ecologists, but learning
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?
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.
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! ). 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.