cutadapt demux-paired demuxes some but not all samples

Hello:
I am attempting to demultiplex paired-end, 300 bp reads from a MiSeq run with dual-indexed data. Barcodes are in the reads. I checked with ‘grep’ and they are the first 8 nt in both R1 and R2 reads. They are not reverse complimented.

I am running qiime2-2020.8 installed with conda on Mac OS X (10.15.6 Catalina) within an Ananconda installation.

From within the qiime2 environment I am calling:

qiime cutadapt demux-paired --i-seqs testdata.qza --m-forward-barcodes-file sample_metadata_gvdh.txt --m-forward-barcodes-column BarcodeForward --m-reverse-barcodes-file sample_metadata_gvdh.txt --m-reverse-barcodes-column BarcodeReverse --o-per-sample-sequences testout.qza --o-untrimmed-sequences untrim.qza --verbose

“testdata” here is the first 1M reads from the FASTQ files so that I could test quickly. I have tried on the full dataset (20M reads) and the results are the same.

I have the forward and reverse barcodes in my metadata file, and I can find them in the sequences using grep. They are not reverse-complimented.

When I run qiime cutadapt demux-paired (as shown above), the command completes successfully, but only demuxes a handful of the 36 samples (see below for output in verbose mode).

This appears possibly similar to a bug report from here:

My collaborator attempted the same thing as me using qiime2 and failed, and they went back to using MacQiime “extract_barcodes.py” to pull the barcodes and create the qza object. However, I do not want to use unsupported legacy software that requires root permission to install. I want to understand what is wrong here.

The warning messages in the below output are because the dual-indexing strategy uses duplicate barcodes within the R1 and R2 reads that together are unique.

Here is the command output:

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:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpaexhr_tw --error-rate 0.1 --minimum-length 1 -o /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.1.fastq.gz --untrimmed-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/forward.fastq.gz --pair-adapters -G file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpid820lh4 -p /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.2.fastq.gz --untrimmed-paired-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/reverse.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/forward.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/reverse.fastq.gz

This is cutadapt 2.10 with Python 3.6.11
Command line parameters: --front file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpaexhr_tw --error-rate 0.1 --minimum-length 1 -o /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.1.fastq.gz --untrimmed-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/forward.fastq.gz --pair-adapters -G file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpid820lh4 -p /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.2.fastq.gz --untrimmed-paired-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/reverse.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/forward.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/reverse.fastq.gz
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACTGTCAC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CATGCGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ATTGGCCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GATCTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘TCAAGCCG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CTGATCCG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘AAGCCCGT’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGCCCG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘TACCCCGT’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CGTGATGA’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACATCGTG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GCCTAAGC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘TGGTCACC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ATTGGCCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GATCTGCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘TCAAGCCG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CTGATCCG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘AAGCCCGT’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GTAGCCCG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘TACCCCGT’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CGTGATGA’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘ACATCGTG’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘GCCTAAGC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘TGGTCACC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter ‘CACTGTCC’ (regular 5’) was specified multiple times! Please make sure that this is what you want.
Processing reads on 1 core in paired-end mode …
[---------=8 ] 00:00:57 250,000 reads @ 228.3 µs/read; 0.26 M reads/minute
Finished in 57.08 s (228 us/read; 0.26 M reads/minute).

=== Summary ===

Total read pairs processed: 250,000
Read 1 with adapter: 6,506 (2.6%)
Read 2 with adapter: 6,506 (2.6%)
Pairs written (passing filters): 0 (0.0%)

Total basepairs processed: 150,056,622 bp
Read 1: 75,184,207 bp
Read 2: 74,872,415 bp
Total written (filtered): 149,894,378 bp (99.9%)
Read 1: 75,131,846 bp
Read 2: 74,762,532 bp

=== First read: Adapter s41.NoRN ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 1842 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 4 3906.2 0 4
4 1 976.6 0 1
7 3 15.3 0 3
8 1832 3.8 0 1832
9 1 3.8 0 1
240 1 3.8 0 1

=== First read: Adapter s42.NoRN ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s43.NoRN ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s44.NoRN ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s45.NoRN ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s46.NoRW ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s47.NoRW ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s48.NoRW ===

Sequence: ACTGTCAC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s49.NoRW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 1353 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 19 3906.2 0 19
4 3 976.6 0 3
6 1 61.0 0 1
7 2 15.3 0 2
8 1326 3.8 0 1326
9 1 3.8 0 1
295 1 3.8 0 1

=== First read: Adapter s50.NoRW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s51.RN ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s52.RN ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s53.RN ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s54.RN ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s55.RW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s56.RW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s57.RW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s58.RW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s59.RW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s60.BMW ===

Sequence: GTAGTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s61.BMW ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 1469 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
4 4 976.6 0 4
5 2 244.1 0 2
7 2 15.3 0 2
8 1461 3.8 0 1461

=== First read: Adapter s62.BMW ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s63.BMW ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s64.BMW ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s65.BMN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s66.BMN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s67.BMN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s68.BMN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s69.BMN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s70.BMTN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s71.BMTN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s72.BMTN ===

Sequence: CATGCGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s73.BMTN ===

Sequence: GACTGTCC; Type: regular 5’; Length: 8; Trimmed: 1842 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 18 3906.2 0 18
4 4 976.6 0 4
5 3 244.1 0 3
7 4 15.3 0 4
8 1810 3.8 0 1810
9 2 3.8 0 2
79 1 3.8 0 1

=== First read: Adapter s74.BMTW ===

Sequence: GACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s75.BMTW ===

Sequence: GACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s76.BMTW ===

Sequence: GACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== First read: Adapter s77.BMTW ===

Sequence: GACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s41.NoRN ===

Sequence: CACTGTCC; Type: regular 5’; Length: 8; Trimmed: 1842 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 10 3906.2 0 10
4 1 976.6 0 1
6 5 61.0 0 5
7 4 15.3 0 4
8 1598 3.8 0 1598
190 3 3.8 0 3
191 18 3.8 0 18
192 16 3.8 0 16
193 3 3.8 0 3
259 3 3.8 0 3
260 2 3.8 0 2
262 1 3.8 0 1
265 9 3.8 0 9
266 126 3.8 0 126
267 1 3.8 0 1
268 7 3.8 0 7
269 26 3.8 0 26
270 9 3.8 0 9

=== Second read: Adapter s42.NoRN ===

Sequence: ATTGGCCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s43.NoRN ===

Sequence: GATCTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s44.NoRN ===

Sequence: TCAAGCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s45.NoRN ===

Sequence: CTGATCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s46.NoRW ===

Sequence: AAGCCCGT; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s47.NoRW ===

Sequence: GTAGCCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s48.NoRW ===

Sequence: TACCCCGT; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s49.NoRW ===

Sequence: CGTGATGA; Type: regular 5’; Length: 8; Trimmed: 1353 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 18 3906.2 0 18
4 2 976.6 0 2
7 2 15.3 0 2
8 1323 3.8 0 1323
9 1 3.8 0 1
89 1 3.8 0 1
104 1 3.8 0 1
131 1 3.8 0 1
167 1 3.8 0 1
197 1 3.8 0 1
202 1 3.8 0 1
270 1 3.8 0 1

=== Second read: Adapter s50.NoRW ===

Sequence: ACATCGTG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s51.RN ===

Sequence: GCCTAAGC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s52.RN ===

Sequence: TGGTCACC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s53.RN ===

Sequence: CACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s54.RN ===

Sequence: ATTGGCCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s55.RW ===

Sequence: GATCTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s56.RW ===

Sequence: TCAAGCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s57.RW ===

Sequence: CTGATCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s58.RW ===

Sequence: AAGCCCGT; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s59.RW ===

Sequence: GTAGCCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s60.BMW ===

Sequence: TACCCCGT; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s61.BMW ===

Sequence: CGTGATGA; Type: regular 5’; Length: 8; Trimmed: 1469 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 11 3906.2 0 11
4 3 976.6 0 3
6 1 61.0 0 1
7 2 15.3 0 2
8 1452 3.8 0 1452

=== Second read: Adapter s62.BMW ===

Sequence: ACATCGTG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s63.BMW ===

Sequence: GCCTAAGC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s64.BMW ===

Sequence: TGGTCACC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s65.BMN ===

Sequence: CACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s66.BMN ===

Sequence: ATTGGCCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s67.BMN ===

Sequence: GATCTGCC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s68.BMN ===

Sequence: TCAAGCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s69.BMN ===

Sequence: CTGATCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s70.BMTN ===

Sequence: AAGCCCGT; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s71.BMTN ===

Sequence: GTAGCCCG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s72.BMTN ===

Sequence: TACCCCGT; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s73.BMTN ===

Sequence: CGTGATGA; Type: regular 5’; Length: 8; Trimmed: 1842 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 12 3906.2 0 12
4 5 976.6 0 5
6 1 61.0 0 1
8 1811 3.8 0 1811
20 1 3.8 0 1
72 1 3.8 0 1
107 1 3.8 0 1
124 1 3.8 0 1
133 1 3.8 0 1
138 1 3.8 0 1
171 1 3.8 0 1
227 1 3.8 0 1
232 1 3.8 0 1
233 1 3.8 0 1
250 1 3.8 0 1
267 1 3.8 0 1
287 1 3.8 0 1

=== Second read: Adapter s74.BMTW ===

Sequence: ACATCGTG; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s75.BMTW ===

Sequence: GCCTAAGC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s76.BMTW ===

Sequence: TGGTCACC; Type: regular 5’; Length: 8; Trimmed: 0 times

=== Second read: Adapter s77.BMTW ===

Sequence: CACTGTCC; Type: regular 5’; Length: 8; Trimmed: 0 times

Welcome to the forum, @mchimenti!
Thanks for submitting such a comprehensive first post. You made looking into this question much easier. :ice_cream:. I think this is the root of the issue:

If I understand you correctly, you’re using a combinatorial dual indexing strategy. I’m pretty sure only Unique Dual Indexing is currently supported in q2-cutadapt. cutadapt itself supports combinatorial dual indexing, and there’s an open issue on the q2-cutadapt repo to support DI, which I suspect means this is planned for the future.

In the meantime, you might try demuxing your data with cutadapt directly, and then importing the demuxed sequences into QIIME 2.