Q2 deblur denoise-other

[arwqiime (ARW)]

Hello, I am experiencing the following error message from ITS1 amplicons sequenced on an Illumina iSeq-100. (16S reads analyzed in parallel could successfully processed by q2)

• Version of QIIME 2: 2019.1 in conda
• What is the exact command or commands you ran? Copy and paste please.
  qiime deblur denoise-other --i-demultiplexed-seqs od2_q-score_AG3-ITS-R1/filtered_sequences.qza --p-trim-length 149 --output-dir od3_deblur_AG3-ITS-R1 --p-jobs-to-start 12 --i-reference-seqs UNITE80-Fun-all-dyn-s-dev.qza

I read in other posts that omitting the --o-p-stats flag would help. Didi it w/ and w/o, no difference.

• What is the exact error message?
  (qiime2-2019.1) q2_AG3-Amplikon1-1 $ more /tmp/qiime2-q2cli-err-1o36821f.log
  Traceback (most recent call last):
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/bin/deblur”, line 684, in
  deblur_cmds()
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/click/core.py”, line 764, in call
  return self.main(*args, **kwargs)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/click/core.py”, line 717, in main
  rv = self.invoke(ctx)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/click/core.py”, line 1137, in invoke
  return _process_result(sub_ctx.command.invoke(sub_ctx))
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/click/core.py”, line 956, in invoke
  return ctx.invoke(self.callback, **ctx.params)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/click/core.py”, line 555, in invoke
  return callback(*args, **kwargs)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/bin/deblur”, line 664, in workflow
  threads=threads_per_sample)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/deblur/workflow.py”, line 320, in remove_artifacts_from_biom_table
  coverage_thresh=coverage_thresh)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/deblur/workflow.py”, line 467, in remove_artifacts_seqs
  if (float(line[2]) >= sim_thresh) and
  IndexError: list index out of range
  Traceback (most recent call last):
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2cli/commands.py”, line 274, in call
  results = action(**arguments)
  File “</home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/decorator.py:decorator-gen-434>”, line 2, in denoise_other
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 231, in bound_callable
  output_types, provenance)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/qiime2/sdk/action.py”, line 365, in callable_executor
  output_views = self._callable(**view_args)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_deblur/_denoise.py”, line 124, in denoise_other
  hashed_feature_ids=hashed_feature_ids)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/site-packages/q2_deblur/_denoise.py”, line 163, in _denoise_helper
  subprocess.run(cmd, check=True)
  File “/home/qiime2/miniconda/envs/qiime2-2019.1/lib/python3.6/subprocess.py”, line 418, in run
  output=stdout, stderr=stderr)
  subprocess.CalledProcessError: Command ‘[‘deblur’, ‘workflow’, ‘–seqs-fp’, ‘/tmp/qiime2-archive-xlfs5icr/92581837-b3ca-4d06-851e-974e6ddf2d34/data’, ‘–output-dir’, ‘/tmp/tmp35wygku1’, ‘–mean-error’, ‘0.005’, ‘–indel-prob’, ‘0.01’, ‘-
  -indel-max’, ‘3’, ‘–trim-length’, ‘149’, ‘–min-reads’, ‘10’, ‘–min-size’, ‘2’, ‘–jobs-to-start’, ‘12’, ‘-w’, ‘–pos-ref-fp’, ‘/tmp/qiime2-archive-gkbgz75j/d23f158e-5c91-41a9-ba60-3d9607ef3dd6/data/dna-sequences.fasta’]’ returned non-
  zero exit status 1.

[Nicholas_Bokulich]

Hi @arwqiime,
This error indicates that something went wrong during the pre-filtering stage. See here for a similar issue/advice to troubleshoot:

Plugin error with Deblur

Hi there, I have imported joined reads to Qiime2 that are about 350 in length and I am trying to use deblur denoise-other. qiime deblur denoise-other –i-demultiplexed-seqs $Outputs/joined_demux.qza –i-reference-seqs $Outputs/rdp_seq_1.qza –p-trim-length 300 –p-min-reads 1 –p-min-size 1 –o-table $Outputs/deblur_table.qza –o-representative-sequences $Outputs/rep_seq_deblur.qza –o-stats $Outputs/deblur_stats.txt –verbose The process seems to run well but then, once all samples …

Some possibilities:

1. The reference database may be inappropriate or there is not enough overlap. You may want to spot-check a few sequences.
2. duplicate sequence IDs were mentioned as a possible culprit in that previous topic. Can you verify that this is not a problem in your data?

How many sequences are in filtered_sequences.qza and what is their length?

[arwqiime (ARW)]

Hi @Nicholas_Bokulich
I changed the UNITE 8.0 reference database from the source files:
‘sh_refs_qiime_ver8_dynamic_s_02.02.2019_dev.fasta’ (Includes singletons set as RefS (in dynamic files))
to
‘sh_refs_qiime_ver8_dynamic_02.02.2019_dev.fasta’ (Includes global and 97% singletons) .

‘qiime deblur denoise-other’ completed now without error messages, but I could not find out, what might be wrong in the ‘s’ source fasta file.

Thank you for directing me to the reference database.