Hello,
I usually run DADA2 in Qiime2 using imported raw sequence files (fastq), but I was just given some outputs from DADA2 ran in R that I want to import and use in Qiime 2. I am having difficulty with the rep-seqs. The DADA2 output that I was given included an .fna file. After looking at the forum, related posts on this topic cover creating the .fna file and then importing. Seeing as I have the .fna file already, I went ahead and imported the file as a .qza. This seemed to work fine, and I was able to create the phylogenetic trees. Here is the code I used up to this point:
qiime tools import
--input-path sv.seqs.fna
--output-path cl_sequences.qza
--type 'FeatureData[Sequence]'
qiime phylogeny align-to-tree-mafft-fasttree
--i-sequences cl_sequences.qza
--o-alignment aligned-rep-seqs.qza
--o-masked-alignment masked-aligned-rep-seqs.qza
--o-tree unrooted-tree.qza
--o-rooted-tree rooted-tree.qza
I also imported the feature table using this code:
qiime tools import
--input-path feature-table2.biom
--type 'FeatureTable[Frequency]'
--input-format BIOMV210Format
--output-path feature-table2.qza
I ran a few basic filtering steps on the table (to remove low frequency features). The feature table looked normal when I ran the summarize command:
qiime feature-table summarize
--i-table feature-table2.qza
--o-visualization feature-table2.qzv
--m-sample-metadata-file sr_metadata.tsv
And I used the feature table to select the minimum sampling depth for the diversity analyses. I ran this command:
qiime diversity core-metrics-phylogenetic
--i-phylogeny rooted-tree.qza
--i-table feature-table2.qza
--p-sampling-depth 25000
--m-metadata-file sr_metadata.tsv
--output-dir core-metrics-results
And I got this error:
Plugin error from diversity:
The table does not appear to be completely represented by the phylogeny.
I re-ran with --verbose:
/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/sklearn/metrics/pairwise.py:1776: DataConversionWarning: Data was converted to boolean for metric jaccard
warnings.warn(msg, DataConversionWarning)
Traceback (most recent call last):
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2cli/commands.py", line 329, in call
results = action(**arguments)
File "", line 2, in core_metrics_phylogenetic
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py", line 244, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py", line 484, in callable_executor
outputs = self._callable(scope.ctx, **view_args)
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_diversity/_core_metrics.py", line 61, in core_metrics_phylogenetic
faith_pd_vector, = faith_pd(table=cr.rarefied_table,
File "", line 2, in faith_pd
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py", line 244, in bound_callable
outputs = self.callable_executor(scope, callable_args,
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/qiime2/sdk/action.py", line 390, in callable_executor
output_views = self._callable(**view_args)
File "", line 2, in faith_pd
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 57, in _disallow_empty_tables
return wrapped_function(*args, **kwargs)
File "/Users/jenniferweinert/miniconda3/envs/qiime2-2021.4/lib/python3.8/site-packages/q2_diversity_lib/alpha.py", line 49, in faith_pd
result = unifrac.faith_pd(table_str, tree_str)
File "unifrac/_api.pyx", line 162, in unifrac._api.faith_pd
ValueError: The table does not appear to be completely represented by the phylogeny.
Plugin error from diversity:
The table does not appear to be completely represented by the phylogeny.
See above for debug info.
Based on what I read on the forum, I understood that this error means that all the features in the table are not in rep-seqs file (at least that is how I interpreted it - correct me if I am wrong!). So I converted the rep-seqs file to a .qzv so I could see what might be missing. I noticed 2 things: 1 - it seems like all the features in the table (and more) are present in the rep-seqs file, at least as far as I can tell. 2 - The rep-seqs.qzv looks different than when I have generated others through DADA2 in Qiime2. So am I missing a step somewhere in converting the .fna file? Or am I completely off-base in my approach to this? Please help!
I have uploaded rep-seqs and feature table .qza files that I generated as well as my metadata file.
cl_sequences.qza (277.0 KB)
feature-table2.qza (13.6 KB)
sr_metadata.tsv (1.3 KB)
Let me know if there is any additional information I need to provide. Thanks!