Hi all,
I’m doing a meta-analysis that includes set of data that were amplified with different set of primers (For example V1-V2, V3-V4). In qiime 1, I had analyzed them using the open reference method, but now I’m a bit lost. Added to this, instead of using OTUs clustering, I want to use the DADA2 generating ASVPreformatted text.
So far I made the following 2 steps
qiime tools import
qiime cutadapt demux-single
And here is my question, what would be the next step to generate ASV from sequences coming from different sets of data?
Should I continue with “qiime dada2 denoise-single” for each set of data and then concatenate the generated files?
Thanks for your help!!
Patricia
Your next step is to used closed-reference based clustering with vsearch against the reference database of your choice. Because you’re dealing with non-overlapping regions, a de novo approach won’t work because the OTUs will all be different and a traditional MAFTT tree won’t cluster them together so you’ll lose in both pythogenetic and non-phylogenetic community structures because there’s not point of comparison. Closed ref clustering at 97% or 99% isn’t perfect; there are things you’ll miss, but it actually puts your samples on a level playing field and means that you can make comparisons.
Hi Justin,
I am very grateful for your quick response. I followed your suggestion, and found that for my case it would be good to use the open_reference method.
But now I have a new problem, I would like to use SILVA as a database, and in particular: silva_132_97_16S.fna
From what I was reading in the forums, the database should not be aligned, and I have to import it to be able to use it in open_reference, but when I import it, it throws me the following error
There was a problem importing /home/patricia/Documentos/Silva_132_release/SILVA_132_QIIME_release/rep_set/rep_set_16S_only/97/silva_132_97_16S.fna:
** /home/patricia/Documentos/Silva_132_release/SILVA_132_QIIME_release/rep_set/rep_set_16S_only/97/silva_132_97_16S.fna is not a (n) QIIME1DemuxFormat file**
Surely I was wrong in choosing the --type option,
What would be the correct way to import this reference database?
Many thanks
It may be better, but it’s still not the best practice because it means that your OTUs are going to be driven by region and so aren’t comparable, like I explained above.
In terms of importing your reference dataset, have you checked out the Training Classifers tutorial? I think your issue is a semantic type one, and I know the tutorials are always help me figure out what semantic types I should be using for imports.