Error: Alpha rarefaction curve after merging the data of two different runs

Technical Support
,
1

Hi!
I am facing a problem in alpha rarefaction curve plotting. In this study my samples were sequenced in two different runs "under same conditions" using Illumina Platform- MiSeq. For that reason I have merged the data from both the runs using same filtering parameters and following DADA2. Till "phylogeny align-to-tree-mafft-fasttree" I am not getting problem.
Also, for Alpha rarefaction curve- I am using the same command

Blockquote

qiime diversity alpha-rarefaction \

--i-table merged-table_SSrun1_2.qza
--i-phylogeny mafft-phylogeny/rooted-tree.qza
--p-max-depth 12352
--m-metadata-file SampleSheet_SS_runs_Merged.tsv
--o-visualization alpha_rarefaction.qzv
--verbose

Blockquote

which I use to work on other analysis but this time it is showing some error
Plugin error from diversity:

Blockquote

Command '['faithpd', '-i', '/tmp/qiime2/rashmi/data/382c0292-0413-40d9-8dab-bbb665619333/data/feature-table.biom', '-t', '/tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk', '-o', '/tmp/q2-AlphaDiversityFormat-71qq3zq2']' returned non-zero exit status 1.

Blockquote

Here is the verbose-base output:

BlockquoteCommand:

faithpd -i /tmp/qiime2/rashmi/data/fb390c27-76cf-4f10-b0fd-40dbfca07064/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-rjqdfbai

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/c2da1f97-fda1-4247-be04-1dc3a0dda91d/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-wyxe2ass

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/27a28358-6a03-4bff-9dda-f76549d6c172/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-_h31bp6m

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/be235a9b-0bcb-4f59-a0d5-e20f972c7aad/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-8oyaaq0z

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/6dce445b-7d98-4015-907d-70a007c3e856/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-1b0viohy

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/2a757a26-bb91-40ed-80e3-940e179fe850/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-qahm81s3

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/35de6704-e06a-467d-9dc1-b6f25c93ece6/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-bfmhbys3

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/ff848797-a422-4385-96b0-d32f890fb572/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-7mgrk33u

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/aeadc0b9-1c84-4250-af28-cd9dbdedd1f0/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-_yf7d_32

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/fa017a6a-e8d8-4cf7-b9b2-df27c88d09ac/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-j39750oo

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command:

faithpd -i /tmp/qiime2/rashmi/data/382c0292-0413-40d9-8dab-bbb665619333/data/feature-table.biom -t /tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk -o /tmp/q2-AlphaDiversityFormat-71qq3zq2

Compute failed in faith_pd_one_off: Table observation IDs are not a subset of the tree tips. This error can also be triggered if a node name contains a single quote (this is unlikely).
Traceback (most recent call last):
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2cli/commands.py", line 468, in call
results = action(**arguments)
File "", line 2, in alpha_rarefaction
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/action.py", line 274, in bound_callable
outputs = self.callable_executor(
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/action.py", line 558, in callable_executor
ret_val = self._callable(output_dir=temp_dir, **view_args)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity/_alpha/_visualizer.py", line 375, in alpha_rarefaction
data = _compute_rarefaction_data(table, min_depth, max_depth,
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity/_alpha/_visualizer.py", line 306, in _compute_rarefaction_data
vector, = alpha_phylo(table=rt, metric=metric,
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/context.py", line 140, in deferred_action
return action_obj._bind(
File "", line 2, in alpha_phylogenetic
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/action.py", line 274, in bound_callable
outputs = self.callable_executor(
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/action.py", line 590, in callable_executor
outputs = self._callable(scope.ctx, **view_args)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity/_alpha/_pipeline.py", line 16, in alpha_phylogenetic
vector, = action(table, phylogeny)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/context.py", line 140, in deferred_action
return action_obj._bind(
File "", line 2, in faith_pd
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/action.py", line 274, in bound_callable
outputs = self.callable_executor(
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/qiime2/sdk/action.py", line 509, in callable_executor
output_views = self._callable(**view_args)
File "", line 2, in faith_pd
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 69, in _disallow_empty_tables
return wrapped_function(*args, **kwargs)
File "", line 2, in faith_pd
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 112, in _validate_requested_cpus
return wrapped_function(*bound_arguments.args, **bound_arguments.kwargs)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity_lib/alpha.py", line 53, in faith_pd
_omp_cmd_wrapper(threads, cmd)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 128, in _omp_cmd_wrapper
return _run_external_cmd(cmd, verbose=verbose, env=env)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/site-packages/q2_diversity_lib/_util.py", line 122, in _run_external_cmd
return subprocess.run(cmd, check=True, env=env)
File "/home/rashmi/miniconda3/envs/qiime2-2023.5/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['faithpd', '-i', '/tmp/qiime2/rashmi/data/382c0292-0413-40d9-8dab-bbb665619333/data/feature-table.biom', '-t', '/tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk', '-o', '/tmp/q2-AlphaDiversityFormat-71qq3zq2']' returned non-zero exit status 1.

Plugin error from diversity:

Command '['faithpd', '-i', '/tmp/qiime2/rashmi/data/382c0292-0413-40d9-8dab-bbb665619333/data/feature-table.biom', '-t', '/tmp/qiime2/rashmi/data/156f2fa1-77ac-4945-8d71-132fd0f13365/data/tree.nwk', '-o', '/tmp/q2-AlphaDiversityFormat-71qq3zq2']' returned non-zero exit status 1.

See above for debug info.

Blockquote

please suggest, how can I resolve this error.
Thanks in advance.

  • Rashmi
2

Hello!

Looks like there are some issues with your tree. Probably some sequence IDs are missing there. How did you obtain rep-seqs.qza? If you merge feature tables from different runs, you should also merge rep-seqs files as well, and then use the merged rep-seqs file to construct a tree. Did you follow the same workflow?

Best,

1 Like
3

Thank you so much for your quick response.

Yes, I have completed that part, too.

Thanks for pointing out

Because, what I think the mistake from my end is that, I had performed filtering for Mitochondria,Eukaryota, and Chloroplast first. Then I have generated the phylogentic tree by using that filtered representative sequence file, while in this case I am using "raw merged table file". May be this is the problem??!!

But which file do you suggest to use while performing Alpha rarefaction? "Only raw merged data without any filtering for any taxa or data after filtering"??/

Thanks again for your help!!!

4

Usually, it is not a problem when some sequences are present in the rep-seqs.qza file but missing in the feature table. But the opposite will cause an error - all features from feature table should be presented in rep-seqs.qza file.

So, you should make sure that all sequences from your feature table are represented in the rep-seqs.qza file that you are using for tree, and then this tree can be used with feature table for further analyses.

5

Dear Timur,

I got your point :+1:.

I will try to re-run the command by using another file that contains the entire piece of information about features.

Thanks a lot for your quick response and help!

Best Regards,

6

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