error in cutting primer with qiime cutadapt trim

User Support
1

I’m running qiime2-2024.2.
I want to trim primers from my reads using this command:

qiime cutadapt trim-paired
--i-demultiplexed-sequences demux.qza
--p-cores 4
--p-front-f CCTACGGGAGGCAGCAG
--p-front-r GGACTACHVGGGTWTCTAAT
--p-error-rate 0.1
--o-trimmed-sequences trimmed-seqs.qza

I get the following error:
Plugin error from cutadapt:

Command '['cutadapt', '--cores', '4', '--error-rate', '0.1', '--times', '1', '--overlap', '3', '--minimum-length', '1', '-q', '0,0', '--quality-base', '33', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-wcwvhz4h/GSC0.3_1_33_L001_R1_001.fastq.gz', '-p', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-wcwvhz4h/GSC0.3_1_93_L001_R2_001.fastq.gz', '--front', 'CCTACGGGAGGCAGCAG--p-front-r', '--front', 'GGACTACHVGGGTWTCTAAT--p-error-rate', '--front', '0.1', '/tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_33_L001_R1_001.fastq.gz', '/tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_93_L001_R2_001.fastq.gz']' returned non-zero exit status 2.

Debug info has been saved to /tmp/qiime2-q2cli-err-5950rea4.log

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

Command: cutadapt --cores 4 --error-rate 0.1 --times 1 --overlap 3 --minimum-length 1 -q 0,0 --quality-base 33 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-_uqcjtwb/GSC0.3_1_33_L001_R1_001.fastq.gz -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-_uqcjtwb/GSC0.3_1_93_L001_R2_001.fastq.gz --front CCTACGGGAGGCAGCAG--p-front-r --front GGACTACHVGGGTWTCTAAT--p-error-rate --front 0.1 /tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_33_L001_R1_001.fastq.gz /tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_93_L001_R2_001.fastq.gz

This is cutadapt 4.6 with Python 3.8.15
Command line parameters: --cores 4 --error-rate 0.1 --times 1 --overlap 3 --minimum-length 1 -q 0,0 --quality-base 33 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-_uqcjtwb/GSC0.3_1_33_L001_R1_001.fastq.gz -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-_uqcjtwb/GSC0.3_1_93_L001_R2_001.fastq.gz --front CCTACGGGAGGCAGCAG--p-front-r --front GGACTACHVGGGTWTCTAAT--p-error-rate --front 0.1 /tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_33_L001_R1_001.fastq.gz /tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_93_L001_R2_001.fastq.gz
ERROR: Character '-' in adapter sequence 'CCTACGGGAGGCAGCAG--P-FRONT-R' is not a valid IUPAC code. Use only characters 'ABCDGHIKMNRSTUVWXY'.
Traceback (most recent call last):
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2cli/commands.py", line 520, in call
results = self._execute_action(
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "", line 2, in trim_paired
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/qiime2/sdk/action.py", line 566, in callable_executor
output_views = self._callable(**view_args)
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_cutadapt/_trim.py", line 229, in trim_paired
run_commands(cmds)
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/site-packages/q2_cutadapt/_trim.py", line 29, in run_commands
subprocess.run(cmd, check=True)
File "/home/wygcsu/miniconda3/envs/qiime2-amplicon-2024.2/lib/python3.8/subprocess.py", line 516, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['cutadapt', '--cores', '4', '--error-rate', '0.1', '--times', '1', '--overlap', '3', '--minimum-length', '1', '-q', '0,0', '--quality-base', '33', '-o', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-_uqcjtwb/GSC0.3_1_33_L001_R1_001.fastq.gz', '-p', '/tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-_uqcjtwb/GSC0.3_1_93_L001_R2_001.fastq.gz', '--front', 'CCTACGGGAGGCAGCAG--p-front-r', '--front', 'GGACTACHVGGGTWTCTAAT--p-error-rate', '--front', '0.1', '/tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_33_L001_R1_001.fastq.gz', '/tmp/qiime2/wygcsu/data/50029cc6-25da-44c4-a2e6-b81378c3821a/data/GSC0.3_1_93_L001_R2_001.fastq.gz']' returned non-zero exit status 2.

2

Hi there @SouthGateProject,

Looks like the issue is "ERROR: Character '-' in adapter sequence 'CCTACGGGAGGCAGCAG--P-FRONT-R' is not a valid IUPAC code. Use only characters 'ABCDGHIKMNRSTUVWXY'."

The error appears to just be a typo where there is no gap between your commands, they are directly next to each other. It's trying to read the '-' at the front of --p-front-r as part of your front adapter sequence from the previous line.

let us know if that helps.

2 Likes
3

Thanks very much! I works well.
But it is very strange. In fact, there is one blank space, it reports error. If two blank space, it is well. I also tried with other data and it is normal even with only one blank space.

2 Likes
4

Perhaps there is a way we can make this more clear for future users!

Can you share with us the commands and examples with one and two blank spaces?

5

very sorry. The error cannot be reproduced. I just tried it again, both is ok.