Ok, this just got weirder…
There was indeed duplicate barcodes (the ones you pointed out). That was my error and I fixed it. I then ran the commands listed in the bash.sh script q2-Osburn10BSS8.All-cutadapt-demux.sh listed below.
#!/bin/bash
#SBATCH -A p31523
#SBATCH -p normal
#SBATCH -t 48:00:00
#SBATCH --mem=48G
#SBATCH -n 4
#SBATCH -N 1
#SBATCH --mail-user=bradley.stevenson@northwestern.edu # change to your email
#SBATCH --mail-type=END
#SBATCH --job-name="import_demux_cutadapt"
#SBATCH --output=%j-%x.out
module purge all
module load python-miniconda3
source activate /software/qiime2/2024.5-amplicon/amplicon-env/
cd /projects/p31523/Osburn10BSAll/ # change to your data directory
OUT_DR=`pwd`/qiime2-8.15.25-out
mkdir -p $OUT_DR
echo "[`date`] Importing data into qiime2 ..."
qiime --version
# import seqs as qza
qiime tools import \
--type MultiplexedPairedEndBarcodeInSequence \
--input-path muxed-pe-barcode-in-seq \
--output-path ${OUT_DR}/multiplexed-seqs.qza
echo "[`date`] Demultiplexing paired-end reads ..."
# demultiplex
qiime cutadapt demux-paired \
--i-seqs ${OUT_DR}/multiplexed-seqs.qza \
--m-forward-barcodes-file Osburn10BSS8.All.tsv \
--m-forward-barcodes-column barcode \
--p-anchor-forward-barcode TRUE\
--p-anchor-reverse-barcode TRUE \
--p-mixed-orientation TRUE\
--o-per-sample-sequences demux.qza \
--o-untrimmed-sequences untrimmed.qza \
--verbose
qiime demux summarize \
--i-data ${OUT_DR}/demux.qza \
--o-visualization ${OUT_DR}/demux.qzv
echo "[`date`] Trimming primer sequences from demultiplexed paired-end reads ..."
# trim forward and reverse primers (515FY-M13/926R Parada primers)
qiime cutadapt trim-paired \
--i-demultiplexed-sequences ${OUT_DR}/demux.qza \
--p-anywhere-f CCGTAAAACGACGGCCAGCCGTGYCAGCMGCCGCGGTAA \
--p-anywhere-r CCGYCAATTYMTTTRAGTTT \
--p-match-read-wildcards \
--p-cores $SLURM_NTASKS \
--o-untrimmed-sequences ${OUT_DR}/demux-untrimmed.qza \
--o-trimmed-sequences ${OUT_DR}/demux-trimmed-no-untrimmed.qza
qiime demux summarize \
--i-data ${OUT_DR}/demux-trimmed-no-untrimmed.qza \
--o-visualization ${OUT_DR}/demux-trimmed-no-untrimmed.qzv
I got the same error, that there were duplicate barcodes but it only lists a single library (EP.LC.03.24.1) this time. Here is the error log…
[Fri Aug 15 14:28:52 CDT 2025] Importing data into qiime2 ...
q2cli version 2024.5.0
Run `qiime info` for more version details.
Imported muxed-pe-barcode-in-seq as MultiplexedPairedEndBarcodeInSequenceDirFmt to /projects/p31523/Osburn10BSAll/qiime2-8.15.25-out/multiplexed-seqs.qza
[Fri Aug 15 14:31:19 CDT 2025] Demultiplexing paired-end reads ...
Traceback (most recent call last):
File "/software/qiime2/2024.5-amplicon/amplicon-env/lib/python3.9/site-packages/q2cli/commands.py", line 520, in __call__
results = self._execute_action(
File "/software/qiime2/2024.5-amplicon/amplicon-env/lib/python3.9/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "<decorator-gen-43>", line 2, in demux_paired
File "/software/qiime2/2024.5-amplicon/amplicon-env/lib/python3.9/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self._callable_executor_(
File "/software/qiime2/2024.5-amplicon/amplicon-env/lib/python3.9/site-packages/qiime2/sdk/action.py", line 576, in _callable_executor_
output_views = self._callable(**view_args)
File "/software/qiime2/2024.5-amplicon/amplicon-env/lib/python3.9/site-packages/q2_cutadapt/_demux.py", line 317, in demux_paired
_check_barcodes_uniqueness(
File "/software/qiime2/2024.5-amplicon/amplicon-env/lib/python3.9/site-packages/q2_cutadapt/_demux.py", line 162, in _check_barcodes_uniqueness
raise ValueError('The following samples have duplicate barcode: %s'
ValueError: The following samples have duplicate barcode: EP.LC.03.24.1
Plugin error from cutadapt:
The following samples have duplicate barcode: EP.LC.03.24.1
See above for debug info.
Usage: qiime demux summarize [OPTIONS]
Summarize counts per sample for all samples, and generate interactive
positional quality plots based on `n` randomly selected sequences.
Inputs:
--i-data ARTIFACT SampleData[SequencesWithQuality |
PairedEndSequencesWithQuality | JoinedSequencesWithQuality]
The demultiplexed sequences to be summarized.
[required]
Parameters:
--p-n INTEGER The number of sequences that should be selected at
random for quality score plots. The quality plots will
present the average positional qualities across all of
the sequences selected. If input sequences are paired
end, plots will be generated for both forward and
reverse reads for the same `n` sequences.
[default: 10000]
Outputs:
--o-visualization VISUALIZATION
[required]
Miscellaneous:
--output-dir PATH Output unspecified results to a directory
--verbose / --quiet Display verbose output to stdout and/or stderr during
execution of this action. Or silence output if
execution is successful (silence is golden).
--example-data PATH Write example data and exit.
--citations Show citations and exit.
--help Show this message and exit.
Examples:
# ### example: demux
qiime demux summarize \
--i-data demux.qza \
--o-visualization visualization.qzv
There was a problem with the command:
(1/1) Invalid value for '--i-data':
/projects/p31523/Osburn10BSAll/qiime2-8.15.25-out/demux.qza does not exist.
[Fri Aug 15 14:33:28 CDT 2025] Trimming primer sequences from demultiplexed paired-end reads ...
Usage: qiime cutadapt trim-paired [OPTIONS]
Search demultiplexed paired-end sequences for adapters and remove them. The
parameter descriptions in this method are adapted from the official cutadapt
docs - please see those docs at https://cutadapt.readthedocs.io for complete
details.
Inputs:
--i-demultiplexed-sequences ARTIFACT
SampleData[PairedEndSequencesWithQuality]
The paired-end sequences to be trimmed. [required]
Parameters:
--p-cores NTHREADS Number of CPU cores to use. [default: 1]
--p-adapter-f TEXT... Sequence of an adapter ligated to the 3' end. The
List[Str] adapter and any subsequent bases are trimmed. If a
`$` is appended, the adapter is only found if it is
at the end of the read. Search in forward read. If
your sequence of interest is "framed" by a 5' and a
3' adapter, use this parameter to define a "linked"
primer - see https://cutadapt.readthedocs.io for
complete details. [optional]
--p-front-f TEXT... Sequence of an adapter ligated to the 5' end. The
List[Str] adapter and any preceding bases are trimmed. Partial
matches at the 5' end are allowed. If a `^`
character is prepended, the adapter is only found if
it is at the beginning of the read. Search in
forward read. [optional]
--p-anywhere-f TEXT... Sequence of an adapter that may be ligated to the
List[Str] 5' or 3' end. Both types of matches as described
under `adapter` and `front` are allowed. If the
first base of the read is part of the match, the
behavior is as with `front`, otherwise as with
`adapter`. This option is mostly for rescuing failed
library preparations - do not use if you know which
end your adapter was ligated to. Search in forward
read. [optional]
--p-adapter-r TEXT... Sequence of an adapter ligated to the 3' end. The
List[Str] adapter and any subsequent bases are trimmed. If a
`$` is appended, the adapter is only found if it is
at the end of the read. Search in reverse read. If
your sequence of interest is "framed" by a 5' and a
3' adapter, use this parameter to define a "linked"
primer - see https://cutadapt.readthedocs.io for
complete details. [optional]
--p-front-r TEXT... Sequence of an adapter ligated to the 5' end. The
List[Str] adapter and any preceding bases are trimmed. Partial
matches at the 5' end are allowed. If a `^`
character is prepended, the adapter is only found if
it is at the beginning of the read. Search in
reverse read. [optional]
--p-anywhere-r TEXT... Sequence of an adapter that may be ligated to the
List[Str] 5' or 3' end. Both types of matches as described
under `adapter` and `front` are allowed. If the
first base of the read is part of the match, the
behavior is as with `front`, otherwise as with
`adapter`. This option is mostly for rescuing failed
library preparations - do not use if you know which
end your adapter was ligated to. Search in reverse
read. [optional]
--p-error-rate PROPORTION Range(0, 1, inclusive_end=True)
Maximum allowed error rate. [default: 0.1]
--p-indels / --p-no-indels
Allow insertions or deletions of bases when
matching adapters. [default: True]
--p-times INTEGER Remove multiple occurrences of an adapter if it is
Range(1, None) repeated, up to `times` times. [default: 1]
--p-overlap INTEGER Require at least `overlap` bases of overlap between
Range(1, None) read and adapter for an adapter to be found.
[default: 3]
--p-match-read-wildcards / --p-no-match-read-wildcards
Interpret IUPAC wildcards (e.g., N) in reads.
[default: False]
--p-match-adapter-wildcards / --p-no-match-adapter-wildcards
Interpret IUPAC wildcards (e.g., N) in adapters.
[default: True]
--p-minimum-length INTEGER
Range(1, None) Discard reads shorter than specified value. Note,
the cutadapt default of 0 has been overridden,
because that value produces empty sequence records.
[default: 1]
--p-discard-untrimmed / --p-no-discard-untrimmed
Discard reads in which no adapter was found.
[default: False]
--p-max-expected-errors NUMBER
Range(0, None) Discard reads that exceed maximum expected
erroneous nucleotides. [optional]
--p-max-n NUMBER Discard reads with more than COUNT N bases. If
Range(0, None) COUNT_or_FRACTION is a number between 0 and 1, it is
interpreted as a fraction of the read length.
[optional]
--p-quality-cutoff-5end INTEGER
Range(0, None) Trim nucleotides with Phred score quality lower
than threshold from 5 prime end. [default: 0]
--p-quality-cutoff-3end INTEGER
Range(0, None) Trim nucleotides with Phred score quality lower
than threshold from 3 prime end. [default: 0]
--p-quality-base INTEGER
Range(0, None) How the Phred score is encoded (33 or 64).
[default: 33]
Outputs:
--o-trimmed-sequences ARTIFACT SampleData[PairedEndSequencesWithQuality]
The resulting trimmed sequences. [required]
Miscellaneous:
--output-dir PATH Output unspecified results to a directory
--verbose / --quiet Display verbose output to stdout and/or stderr
during execution of this action. Or silence output
if execution is successful (silence is golden).
--example-data PATH Write example data and exit.
--citations Show citations and exit.
--help Show this message and exit.
There was a problem with the command:
(1/1?) No such option: --o-untrimmed-sequences Did you mean --o-trimmed-
sequences?
Usage: qiime demux summarize [OPTIONS]
Summarize counts per sample for all samples, and generate interactive
positional quality plots based on `n` randomly selected sequences.
Inputs:
--i-data ARTIFACT SampleData[SequencesWithQuality |
PairedEndSequencesWithQuality | JoinedSequencesWithQuality]
The demultiplexed sequences to be summarized.
[required]
Parameters:
--p-n INTEGER The number of sequences that should be selected at
random for quality score plots. The quality plots will
present the average positional qualities across all of
the sequences selected. If input sequences are paired
end, plots will be generated for both forward and
reverse reads for the same `n` sequences.
[default: 10000]
Outputs:
--o-visualization VISUALIZATION
[required]
Miscellaneous:
--output-dir PATH Output unspecified results to a directory
--verbose / --quiet Display verbose output to stdout and/or stderr during
execution of this action. Or silence output if
execution is successful (silence is golden).
--example-data PATH Write example data and exit.
--citations Show citations and exit.
--help Show this message and exit.
Examples:
# ### example: demux
qiime demux summarize \
--i-data demux.qza \
--o-visualization visualization.qzv
There was a problem with the command:
(1/1) Invalid value for '--i-data':
/projects/p31523/Osburn10BSAll/qiime2-8.15.25-out/demux-trimmed-no-
untrimmed.qza does not exist.
I have uploaded the metadata file (Osburn10BSS8.All.tsv). I am still stumped.
Thanks for any insights!
Osburn10BSS8.All.tsv (14.2 KB)
Brad
