Some help is needed, I’m running qiime2-2019.7 through conda and am having problems with the vsearch plugin, I have some paired end samples of the 16s v3v4 region (341f - 806r), which I successfully trimmed primers/barcodes with the Cutadapt plugin and denoised/derep/merged with the dada2 plugin, and successfully trained the Greengene 13_8 97% reference with the primer and got taxonmy of good resolution(using q2-classifier), however, when I was using the vsearch plugin to produce close-referenced results compatible with Faprotax/Bugbase, I ran in to problems. Here are the commands which are not working:
qiime vsearch cluster-features-closed-reference \
it returned with:
Plugin error from vsearch:
No matches were identified to reference_sequences. This can happen if sequences are not homologous to reference_sequences, or if sequences are not in the same orientation as reference_sequences (i.e., if sequences are reverse complemented with respect to reference sequences). Sequence orientation can be adjusted with the strand parameter.
Which is really weird since I used the exact same reference sequences to train ref-seqs and already got good results, I tried changing the ref-seqs used here to the ones I trained for my primers and still got the same error, I tried lowering the perc-identity from 0.99 to 0.95, 0.9, 0.85 etc. all the way to 0.70 and yet the error persists, can anyone tell me what seems to be the problem? I used to run the exact same procedure with other data with no problem, I suspect the low quality of this particular data (due to the long sequencing range of the primer, around 500bp) is causing the problem but lowering perc-identity is not working as well, I also tried 99%, 94% and 91% from the gg 16s database but still no,table-dada2.qza (157.0 KB) rep-seqs-dada2.qza (447.5 KB) I uploaded the table and the sequence output from dada2 here, but the gg reference is too large, but its a standard gg 13_8 16s fasta data imported to q2 as follow:
qiime tools import **
–input-path 97_otus.fasta **
–output-path gg_97_otus.qza **
and was used before for the same purpose with no problem. Any help is greatly appreciated, thanks!!