Wierd error when I used cutadpt plugin

I have got this error, while I fully completed other libraries. Analysing with this current library gives such an error! I could not solve it.

Hi @TurboQiimer, we need much more information in order to effectively help you. Please provide us with:

  • The version of QIIME 2 used
  • The complete error observed - copy-and-paste when running the command with the --verbose flag

:qiime2:

Thanks for the fast reply!

The version is: 2020/8

The complete error:

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: cutadapt --front file:/tmp/tmpykhw4nae --error-rate 0.1 --minimum-length 1 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.1.fastq.gz --untrimmed-output /tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/forward.fastq.gz --pair-adapters -G file:/tmp/tmpmrqxs_o3 -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.2.fastq.gz --untrimmed-paired-output /tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/reverse.fastq.gz /tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/forward.fastq.gz /tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/reverse.fastq.gz

This is cutadapt 2.10 with Python 3.6.10
Command line parameters: --front file:/tmp/tmpykhw4nae --error-rate 0.1 --minimum-length 1 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.1.fastq.gz --untrimmed-output /tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/forward.fastq.gz --pair-adapters -G file:/tmp/tmpmrqxs_o3 -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.2.fastq.gz --untrimmed-paired-output /tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/reverse.fastq.gz /tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/forward.fastq.gz /tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/reverse.fastq.gz
Processing reads on 1 core in paired-end mode ...
cutadapt: error: Error in sequence file at unknown line: Reads are improperly paired. Read name 'SN863:637:HFHFNBCX3:1:1104:2167:2226 1:N:0:TCCTCAAT' in file 1 does not match 'SN863:637:HFHFNBCX3:2:1101:1191:2126 1:N:0:TCCTCAAT' in file 2.
Traceback (most recent call last):
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2cli/commands.py", line 329, in __call__
    results = action(**arguments)
  File "<decorator-gen-522>", line 2, in demux_paired
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
    output_types, provenance)
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/qiime2/sdk/action.py", line 390, in _callable_executor_
    output_views = self._callable(**view_args)
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2_cutadapt/_demux.py", line 213, in demux_paired
    error_rate, mux_fmt, batch_size, minimum_length)
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2_cutadapt/_demux.py", line 164, in _demux
    run_command(cmd)
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/site-packages/q2_cutadapt/_demux.py", line 36, in run_command
    subprocess.run(cmd, check=True)
  File "/home/mpi/miniconda3/envs/qiime2-2020.8/lib/python3.6/subprocess.py", line 438, in run
    output=stdout, stderr=stderr)
subprocess.CalledProcessError: Command '['cutadapt', '--front', 'file:/tmp/tmpykhw4nae', '--error-rate', '0.1', '--minimum-length', '1', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.1.fastq.gz', '--untrimmed-output', '/tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/forward.fastq.gz', '--pair-adapters', '-G', 'file:/tmp/tmpmrqxs_o3', '-p', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.2.fastq.gz', '--untrimmed-paired-output', '/tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/reverse.fastq.gz', '/tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/forward.fastq.gz', '/tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/reverse.fastq.gz']' returned non-zero exit status 1.

Plugin error from cutadapt:

  Command '['cutadapt', '--front', 'file:/tmp/tmpykhw4nae', '--error-rate', '0.1', '--minimum-length', '1', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.1.fastq.gz', '--untrimmed-output', '/tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/forward.fastq.gz', '--pair-adapters', '-G', 'file:/tmp/tmpmrqxs_o3', '-p', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-7h5x1ar4/{name}.2.fastq.gz', '--untrimmed-paired-output', '/tmp/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-munhdl5j/reverse.fastq.gz', '/tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/forward.fastq.gz', '/tmp/qiime2-archive-kavfqa92/d9fc4320-051d-442e-bc9f-f50bb6492517/data/reverse.fastq.gz']' returned non-zero exit status 1.

See above for debug info.

supplementary info:
the command used.

qiime cutadapt demux-paired \
> --i-seqs B1.qza \
> --m-forward-barcodes-file dsrbmetadata.tsv \
> --m-forward-barcodes-column barcodeF \
> --m-reverse-barcodes-file dsrbmetadata.tsv \
> --m-reverse-barcodes-column barcodeR \
> --o-per-sample-sequences demux_paired.gza \
> --o-untrimmed-sequences untrimmed.gza \
> --verbose

Thanks a lot.

Hello @TurboQiimer,
The clue to the error lies in this line

These read names do not match and therefore Cutadapt is throwing an error. I would make sure that all the read name of the sequences you are trying to join are identical and then run the command again. Once the read names line up you should be good to go!

I hope this helps :qiime2:

Chloe

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