Thank you very much for providing me with the correction - I appreciate it. I was able to import the taxonomy data.
I am working through the rest of what I need to do in order re-analyze my data with QIIME 2. In QIIME 1 - I ran open reference OTU picking. I was able to successfully run this in QIIME 2 with VSearch but the QIIME 1 script wrapped many of the steps / tools - clustering, alignment, classification and generating a phylogenetic tree with the one pick_open_reference_otus.py script. I am trying to repeat what I did in QIIME 1 but I need a little help in making sure I do everything in the correct order and with the proper output files from each preceding step.
Open Reference OTU picking with VSearch:
`(qiime2-2017.12) bash-3.2$ qiime vsearch cluster-features-open-reference --i-sequences ./20180127_pilot_rep-seqs-denovo_and_ref_nonchimeric.qza --i-table pilot_chimera_filtered_table.qza --i-reference-sequences ../QIIME2/SILVA_16S/99_otus.qza --p-perc-identity 0.99 --p-threads 6 --o-clustered-table ./20180129_pilot_VSearch_OpenRef_99per_Clustered_Table.qza --o-clustered-sequences 20180129_pilot_VSearch_OpenRef_99per_Clustered_Seqs.qza --o-new-reference-sequences 20180129_pilot_VSearch_OpenRef_99per_New_Ref_Seqs.qza --verbose`
Saved FeatureTable[Frequency] to: ./20180129_pilot_VSearch_OpenRef_99per_Clustered_Table.qza
Saved FeatureData[Sequence] to: 20180129_pilot_VSearch_OpenRef_99per_Clustered_Seqs.qza
Saved FeatureData[Sequence] to: 20180129_pilot_VSearch_OpenRef_99per_New_Ref_Seqs.qza
I am not quite certain what the difference is between the two output sequence artifact files. Could you possibly clarify? Which one is to be used for the next step in the analysis process - the clustered seqs or the new reference seqs?
In QIIME 1 Open Reference OTU picking classification of the clustered OTUs was the next step but in the “Moving Pictures Tutorial” it is listed much later - after diversity analyses. Wouldn’t I want to repeat the next steps of analysis for open reference OTU picking in the following order:
Classify Clustered Seqs / OTUs - Includes training the classifier with the Silva reference sequences and taxonomy.
Alignment of sequences - MAFFT
Generate phylogenetic tree with aligned sequences
(3.5: I normally filtered my biom file output from the pick_open_reference_otus.py script based on OTUs that were taxonomically unassigned or I would remove other OTUs or samples based on my review.) - but I am still figuring out when do these types of filtering with QIIME 2)
- Complete Alpha and Beta Diversity Analyses
Thank you again for your time and help with this. I could not find a full Open-Reference OTU Picking tutorial / pipeline suggestion. I am trying to replicate most of what I did in QIIME 1.
Thank you very much,