dada2 denoise-paired error using qiime 2019.4

Dear Friends,

When running dada2 on paired end DNA data:

qiime dada2 denoise-paired --i-demultiplexed-seqs ww-DNA_1to11_paired-end-demux-trimmed-2.qza --p-trunc-len-f 301 --p-trunc-len-r 301 --p-trim-left-f 0 --p-trim-left-r 0 --p-chimera-method consensus --p-n-threads 10 --o-representative-sequences ww-DNA_1to11_paired-end-demux-trimmed_dada2-rep-seqs.qza --o-table ww-DNA_1to11_paired-end-demux-trimmed-dada2-rep-seqs-table.qza --o-denoising-stats ww-DNA_1to11_paired-end-demux-trimmed_dada2-rep-seqs-stats.qza

I get this error:

Running external command line application(s). This may print messages to stdout and/or stderr.
The command(s) being run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.

Command: run_dada_paired.R /data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/forward /data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/reverse /data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/output.tsv.biom /data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/track.tsv /data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/filt_f /data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/filt_r 301 301 0 0 2.0 2 consensus 1.0 10 1000000

R version 3.5.1 (2018-07-02)
Loading required package: Rcpp
DADA2: 1.10.0 / Rcpp: 1.0.1 / RcppParallel: 4.4.2

  1. Filtering ..........
  2. Learning Error Rates
    14749 total bases in 49 reads from 10 samples will be used for learning the error rates.
    14749 total bases in 49 reads from 10 samples will be used for learning the error rates.
  3. Denoise remaining samples ..........
  4. Remove chimeras (method = consensus)
    Error in isBimeraDenovoTable(unqs[[i]], ..., verbose = verbose) :
    Input must be a valid sequence table.
    Calls: removeBimeraDenovo -> isBimeraDenovoTable
    Execution halted
    Traceback (most recent call last):
    File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 231, in denoise_paired
    run_commands([cmd])
    File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
    subprocess.run(cmd, check=True)
    File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/subprocess.py", line 418, in run
    output=stdout, stderr=stderr)
    subprocess.CalledProcessError: Command '['run_dada_paired.R', '/data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/forward', '/data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/reverse', '/data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/output.tsv.biom', '/data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/track.tsv', '/data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/filt_f', '/data/deepak-data/NPRP-11-data/qiime-results/qiime-tmp/tmpyk1qtmtq/filt_r', '301', '301', '0', '0', '2.0', '2', 'consensus', '1.0', '10', '1000000']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/q2cli/commands.py", line 311, in call
results = action(**arguments)
File "</root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/decorator.py:decorator-gen-451>", line 2, in denoise_paired
File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py", line 231, in bound_callable
output_types, provenance)
File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/qiime2/sdk/action.py", line 365, in callable_executor
output_views = self._callable(**view_args)
File "/root/miniconda2/envs/qiime2-2019.4/lib/python3.6/site-packages/q2_dada2/_denoise.py", line 246, in denoise_paired
" and stderr to learn more." % e.returncode)
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

Plugin error from dada2:

An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

See above for debug info.

Attached is my trimmed qzv file from which primers were removed. Could you please let me know what could be the issue here? Thanks for your time.

ww-DNA_1to11_paired-end-demux-trimmed-2.qzv (304.2 KB)

Hi @danielsebas!

Yes indeed, right this way:

This error happens when your DADA2 is unable to join any of your reads. It is possible that your inclusion of so many low-quality nt positions is causing this. Any chance you can tighten up your trim/trunc params? This will depend on the length of the target region in question. Another option is to also proceed with only the forward reads.

:qiime2: :t_rex:

1 Like

Yes, I removed the bad nts based on quality score and it ran well :slight_smile:

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