Plugin error from cutadapt: when running Demultiplexing with dual barcodes...

[Pratchaya]

Hello,

I have a problem that I could not solve it by myself. Previously I did not have this problem.

After running this following code:

=== Step 2: Demultiplexing ===

echo "Step 2: Demultiplexing with dual barcodes..."
qiime cutadapt demux-paired
--i-seqs "/netscratch/dep_psl/grp_hacquard/Pratchaya/Amplicons/PP_5th_042026/tmp_16S/PP01/demux/multiplexed-seqs.qza"
--p-error-rate 0.17
--m-forward-barcodes-file $map
--m-reverse-barcodes-file $map
--m-forward-barcodes-column Forward_Barcode
--m-reverse-barcodes-column Reverse_Barcode
--o-per-sample-sequences "/netscratch/dep_psl/grp_hacquard/Pratchaya/Amplicons/PP_5th_042026/tmp_16S/PP01/processed/demultiplexed-seqs.qza"
--o-untrimmed-sequences "/netscratch/dep_psl/grp_hacquard/Pratchaya/Amplicons/PP_5th_042026/tmp_16S/PP01/processed/untrimmed-seqs.qza"
--p-mixed-orientation TRUE
--p-cores 32
qiime demux summarize
--i-data "/netscratch/dep_psl/grp_hacquard/Pratchaya/Amplicons/PP_5th_042026/tmp_16S/PP01/processed/demultiplexed-seqs.qza"
--o-visualization "/netscratch/dep_psl/grp_hacquard/Pratchaya/Amplicons/PP_5th_042026/tmp_16S/PP01/processed/demultiplexed-seqs.qzv"

where the directory containing the raw data: rawdata="/netscratch/dep_psl/grp_hacquard/Pratchaya/Amplicons/PP_5th_042026/raw_16S_combined/PP01/"

rawdata contains forward.fastq.gz and reverse.fastq.gz

It gives me this error:

Command '['cutadapt', '--front', 'file:/scratch/tmp0weqzbek', '--error-rate', '0.17', '--minimum-length', '1', '-o', '/scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.1.fastq.gz', '--untrimmed-output', '/scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/forward.fastq.gz', '--pair-adapters', '-G', 'file:/scratch/tmpsk0cvrll', '-p', '/scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.2.fastq.gz', '--untrimmed-paired-output', '/scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/reverse.fastq.gz', '/scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/forward.fastq.gz', '/scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/reverse.fastq.gz', '-j', '32']' returned non-zero exit status 1.

Debug info has been saved to /scratch/qiime2-q2cli-err-3pcm4agi.log

Could someone please help me ?

Thank you very much in advance for your time!

With best regards,

Pratchaya

[pmomocabr (Paula Momo)]

Hi Pratchaya!

Welcome to the forum:)

To have a bit more context of the possible error, would you mind sharing the content of your cat /scratch/qiime2-q2cli-err-3pcm4agi.log by eg. cat /scratch/qiime2-q2cli-err-3pcm4agi.log?

Thank you!

Paula

[Pratchaya]

@pmomocabr

cat /scratch/qiime2-q2cli-err-3pcm4agi.loHi

Hi Paula,

Thank you very much for your time.

Yes, please have a check at the following, I am sorry in advance that the text is too long:

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:/scratch/tmp0weqzbek --error-rate 0.17 --minimum-length 1 -o /scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.1.fastq.gz --untrimmed-output /scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/forward.fastq.gz --pair-adapters -G file:/scratch/tmpsk0cvrll -p /scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.2.fastq.gz --untrimmed-paired-output /scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/reverse.fastq.gz /scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/forward.fastq.gz /scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/reverse.fastq.gz -j 32

This is cutadapt 4.5 with Python 3.8.15

Command line parameters: --front file:/scratch/tmp0weqzbek --error-rate 0.17 --minimum-length 1 -o /scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.1.fastq.gz --untrimmed-output /scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/forward.fastq.gz --pair-adapters -G file:/scratch/tmpsk0cvrll -p /scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.2.fastq.gz --untrimmed-paired-output /scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/reverse.fastq.gz /scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/forward.fastq.gz /scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/reverse.fastq.gz -j 32

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATTTAGAGGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGGCATCTAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCCAACGGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCCCTTGTCTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATCCTTTGGTTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TACGAGCCCTAA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGTGTTACTCCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TATCGCGCGATA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTAGAGCTCCCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGGATCTAGTGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CCAGATATAGCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AATAGCATGTCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCATTCCACTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGCCAGTCATAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTGTCGATTCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACGAGACTGATT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTCGAACGAGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGTCGCAAATAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGCACAGTCGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTCTACGAACAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CACGCAGTCTAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTCTCCATCACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GGTCTCCTACAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AAGTCACACACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACCATCCAACGA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GAGTTAGCATCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCCGAGTCACCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACCGGTATGTAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACCAGTGACTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAATAGACACC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTCTAGAGTGCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTCCTCCCTTAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAAACATGGTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTTAGGACGAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAGCTCAAACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CACACAAAGTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATGCCGGTAATA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TAAGACTACTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTCTGTCTGCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGCATACACTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CCAATACGCCTG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCTCTACCACTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACACCTGCGATC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGTGTTATGTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCGAAACATGCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GGTTTAACACGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATAGCGAACTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCTCCATTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCAACCCGTGAA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTCTCCTCCCTT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGACAAGCTTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGGTCAACGATA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GCAACACCATCC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGATCGAACACT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTCCTCTCCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTAGCAGCGTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTCCCACCCATT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGACAGTAGGAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AACCGCATAAGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CATCAAGCATAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCTGTAGAGCCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTTAGCTACTCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATCTAATCGAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATCGCACAGTAA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTCGTGCACAT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTGCAAGCAAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CATCGACGAGTT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CAAGTTTCCGCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGCAGAACATCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GCAGATTTCCAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTTGAGAAATCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CAAGCCCTAGTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCGGATCTGTGA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATCGAATCGAGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TAAACGCGACTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTCGTGTAGCCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTTACGAGCTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGCGCTCACATC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CACCACAGAATC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CCTTGTTCACCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GAAACATCCCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGATGATCAGTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CAGTCGTTAAGA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CCTCTGAGAGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACTACCTCTTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGCGTCAAACTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCACGAGTCACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TACAGCGCATAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTGCGTTAGCAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCGACCAAACAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GGTCTTAGCACC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AACCAGCAGATT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTGTCAGTGACC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TATCACCGGCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTTCCAACTCAT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAAGAACCTTG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTATCCAAGTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATCAACCCACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTGAACAAGCCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCCCTTGTCTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATCCTTTGGTTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TACGAGCCCTAA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGTGTTACTCCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TATCGCGCGATA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTAGAGCTCCCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGGATCTAGTGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CCAGATATAGCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AATAGCATGTCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCATTCCACTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGCCAGTCATAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTGTCGATTCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACGAGACTGATT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTCGAACGAGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGTCGCAAATAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGCACAGTCGCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTCTACGAACAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CACGCAGTCTAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTCTCCATCACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GGTCTCCTACAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AAGTCACACACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACCATCCAACGA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GAGTTAGCATCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCCGAGTCACCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACCGGTATGTAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACCAGTGACTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAATAGACACC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTCTAGAGTGCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTCCTCCCTTAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAAACATGGTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTTAGGACGAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACAGCTCAAACA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CACACAAAGTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATGCCGGTAATA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TAAGACTACTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTCTGTCTGCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGCATACACTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CCAATACGCCTG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCTCTACCACTC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ACACCTGCGATC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGTGTTATGTGG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCGAAACATGCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GGTTTAACACGC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATAGCGAACTCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTTCCTCCATTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCAACCCGTGAA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GTCTCCTCCCTT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGACAAGCTTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TGGTCAACGATA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GCAACACCATCC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CGATCGAACACT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTCCTCTCCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTAGCAGCGTA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TTCCCACCCATT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGACAGTAGGAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AACCGCATAAGT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CATCAAGCATAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'TCTGTAGAGCCA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CTTAGCTACTCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GATCTAATCGAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATCGCACAGTAA' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGTCGTGCACAT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'ATTGCAAGCAAC' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CATCGACGAGTT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'CAAGTTTCCGCG' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'AGCAGAACATCT' (regular 5') was specified multiple times! Please make sure that this is what you want.

WARNING: Adapter 'GCAGATTTCCAG' (regular 5') was specified multiple times! Please make sure that this is what you want.

Processing paired-end reads on 32 cores ...

ERROR: [Errno 24] Too many open files

Traceback (most recent call last):

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/q2cli/commands.py", line 520, in _call_

results = self.\_execute_action(

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/q2cli/commands.py", line 581, in _execute_action

results = action(\*\*arguments)

File "", line 2, in demux_paired

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable

outputs = self.\_callable_executor\_(

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/qiime2/sdk/action.py", line 566, in _callable_executor_

output_views = self.\_callable(\*\*view_args)

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/q2_cutadapt/_demux.py", line 273, in demux_paired

untrimmed = \_demux(

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/q2_cutadapt/_demux.py", line 224, in _demux

run_command(cmd)

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/site-packages/q2_cutadapt/_demux.py", line 38, in run_command

subprocess.run(cmd, check=True)

File "/netscratch/dep_psl/common/novogene_pipeline/conda/lib/python3.8/subprocess.py", line 516, in run

raise CalledProcessError(retcode, process.args,

subprocess.CalledProcessError: Command '['cutadapt', '--front', 'file:/scratch/tmp0weqzbek', '--error-rate', '0.17', '--minimum-length', '1', '-o', '/scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.1.fastq.gz', '--untrimmed-output', '/scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/forward.fastq.gz', '--pair-adapters', '-G', 'file:/scratch/tmpsk0cvrll', '-p', '/scratch/q2-CasavaOneEightSingleLanePerSampleDirFmt-o_endi2g/{name}.2.fastq.gz', '--untrimmed-paired-output', '/scratch/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-3gj11ygi/reverse.fastq.gz', '/scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/forward.fastq.gz', '/scratch/qiime2/ppramoj/data/3d27a8c2-8b14-43bb-b4d7-c2e906e00d26/data/reverse.fastq.gz', '-j', '32']' returned non-zero exit status 1

Thank you very much.

Please let me know.

With best regards,

Pratchaya

[pmomocabr (Paula Momo)]

Hi Pratchaya

Thanks for sharing the log! We can start with two things I can see going on for now:

1. "Too many files error": ERROR: [Errno 24] Too many open files This looks like a system resource limit being hit when cutadapt tries to run with 32 cores and many samples at once. You could try reducing --p-cores 32 to eg. --p-cores 4 and rerun and see if this already solves the issue.

2. Could be worth having a look at the mapping file: Some barcodes appear hundreds of times in the warnings, which I think it could be ok depending on the indexing strategy that was used, but just in case could you check if the mapping file has duplicate rows? Eg. wc -l $map and verify the number of lines correspond to your expected number of samples?

Let me know what you find!

Paula

[Pratchaya]

Hi Paula,

Thank you very much for your suggestions!

I am working on it now.

I am going to let you know.

Have a good day.

Thank you.

With best regards,

Pratchaya