Deblur Denoise Plugin Error Message

Hello- I am running qiime2-amplicon-2024.5
I am trying to run deblur denoise on CO1 sequence data and continue to get this error message

"Plugin error from deblur:

Command '['deblur', 'workflow', '--seqs-fp', '/tmp/qiime2/ajanosik/data/f4ed7265-dce0-4d25-9226-57540e4e29f0/data', '--output-dir', '/tmp/tmpnyxqf7dq', '--mean-error', '0.005', '--indel-prob', '0.01', '--indel-max', '3', '--trim-length', '230', '--left-trim-length', '50', '--min-reads', '10', '--min-size', '2', '--jobs-to-start', '8', '-w', '--pos-ref-fp', '/tmp/qiime2/ajanosik/data/9a769ac4-1d70-4fb9-9147-d42690c59091/data/dna-sequences.fasta']' returned non-zero exit status 1.

Debug info has been saved to /tmp/qiime2-q2cli-err-6a70l5ai.log"

These are the commands I have ran so far-
qiime tools import --type 'SampleData[SequencesWithQuality]'
--input-path /home/users/ajanosik/Jake_Roush/Full_Manifest.txt
--output-path single-end-demux.qza \
--input-format SingleEndFastqManifestPhred33V2

qiime quality-filter q-score
--i-demux single-end-demux.qza
--o-filtered-sequences single-end-demux-filtered.qza
--o-filter-stats single-end-demux-stats.qza

qiime deblur denoise-other
--i-demultiplexed-seqs single-end-demux-filtered.qza
--i-reference-seqs /home/users/ajanosik/Jake_Roush/References/reference_sequence_trim2.qza
--p-trim-length 230 \ (Tried 238,230,200 and it gave a "deblur plugin error" message
--p-left-trim-len 50\ (Tried trimming from the left to see if it work work also)
--o-representative-sequences rep-seqs.qza
--o-table table.qza
--p-sample-stats \ (I have tried command without this, incase memory storage was issue.)
--o-stats deblur-stats.qza
--p-jobs-to-start 8

When I ran the debug info this is what got kicked back- cat /tmp/qiime2-q2cli-err-6a70l5ai.log

/home/share/anaconda/envs/qiime2-amplicon-2024.5/bin/deblur:534: DeprecationWarning: The 'warn' method is deprecated, use 'warning' instead
logger.warn('deblur version %s workflow started on %s' %
/home/share/anaconda/envs/qiime2-amplicon-2024.5/bin/deblur:536: DeprecationWarning: The 'warn' method is deprecated, use 'warning' instead
logger.warn('parameters: %s' % locals())
Traceback (most recent call last):
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/bin/deblur", line 687, in
deblur_cmds()
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/click/core.py", line 1157, in call
return self.main(*args, **kwargs)
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/click/core.py", line 1078, in main
rv = self.invoke(ctx)
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/click/core.py", line 1688, in invoke
return _process_result(sub_ctx.command.invoke(sub_ctx))
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/click/core.py", line 1434, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/click/core.py", line 783, in invoke
return __callback(*args, **kwargs)
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/bin/deblur", line 664, in workflow
tmp_files = remove_artifacts_from_biom_table(output_fp, outputfasta_fp,
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/deblur/workflow.py", line 316, in remove_artifacts_from_biom_table
clean_fp, num_seqs_left, tmp_files = remove_artifacts_seqs(
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/deblur/workflow.py", line 466, in remove_artifacts_seqs
if (float(line[2]) >= sim_thresh) and
IndexError: list index out of range
Traceback (most recent call last):
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 520, in call
results = self._execute_action(
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "", line 2, in denoise_other
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 576, in callable_executor
output_views = self._callable(**view_args)
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_deblur/_denoise.py", line 120, in denoise_other
return _denoise_helper(
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_deblur/_denoise.py", line 185, in _denoise_helper
subprocess.run(cmd, check=True)
File "/home/share/anaconda/envs/qiime2-amplicon-2024.5/lib/python3.9/subprocess.py", line 528, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['deblur', 'workflow', '--seqs-fp', '/tmp/qiime2/ajanosik/data/f4ed7265-dce0-4d25-9226-57540e4e29f0/data', '--output-dir', '/tmp/tmpnyxqf7dq', '--mean-error', '0.005', '--indel-prob', '0.01', '--indel-max', '3', '--trim-length', '230', '--left-trim-length', '50', '--min-reads', '10', '--min-size', '2', '--jobs-to-start', '8', '-w', '--pos-ref-fp', '/tmp/qiime2/ajanosik/data/9a769ac4-1d70-4fb9-9147-d42690c59091/data/dna-sequences.fasta']' returned non-zero exit status 1.

I'm not sure how to interpret this or what my next steps should be. Please help me in fixing this.
Thank you!

Hi @jakeroush4,

I would hazard a guess that this might be a memory issue? Can you provide more details on the computer system you're running?, i.e. RAM, CPU, Storage?

Also how large is your reference_sequence_trim2.qza file?

Based on my understanding of deblur, you do not necessarily need the full reference sequence file for denoising. You only need enough reference sequences for deblur to accurately determine if your amplicon sequences are in fact CO1 sequences.

If this reference file is too large perhaps make use of qiime rescript subsample-fasta ... to randomly subset the reference_sequence_trim2.qza file and then use that as input to --i-reference-seqs.

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