Problems with demultiplexing dual-indexed pair-end sequences from MiSeq, Illumina

[Viktoria]

Hello! I have multiplexed data from MiSeq, Illumina - 2 fastq.gz files (forward and reverse reads). All my samples are dual-indexed (the same forward barcode but different reverse barcodes) so I create 2 metadata files (Forward.tsv and Reverse.tsv). I check them with Keemei from Google Sheets. They are correct.

I use qiime2-2020.8 version from conda environment with Ubuntu 20.04 terminal.

I create .qza file from forward.fastq.gz and reverse.fastq.gz files with this command:
qiime tools import --type MultiplexedPairedEndBarcodeInSequence --input-path ~/Документы/qiime --output-path paired.qza

Then I choose "cutadapt demux-paired" command for demultiplexing. Am I correct?

I run the command:
qiime cutadapt demux-paired --i-seqs paired.qza --m-forward-barcodes-file Forward.tsv --m-forward-barcodes-column seq --m-reverse-barcodes-file Reverse.tsv --m-reverse-barcodes-column seq --o-per-sample-sequences demux-f-r.qza --o-untrimmed-sequences untrim-f-r.qza

But it ends with the error:
cutadapt: error: Character 'P' in adapter sequence 'SAMPLEID2-ERY-SAND CCTTTATAGTCC2-ERY-STM CCTTTATAGTCC1-SAND CCTTTATAGTCC3-SAND CCTTTATAGTCC5-SAND CCTTTATAGTCC1-STM CCTTTATAGTCC3-STM CCTTTATAGTCC5-STM CCTTTATAGTCCMIN-1 CCTTTATAGTCCMIN-2 CCTTTATAGTCCNAME: SEQ, DTYPE: OBJECT' is not a valid IUPAC code. Use only characters XACGTURYSWKMBDHVN.

I can't find this error in my data. What should I do?
Thanks!

Metadata with forward barcodes

Metadata with reverse barcodes

[Viktoria]

I tried to use one metadata file

and the command ended without errors but I received the strange result

My commands were:

qiime cutadapt demux-paired --i-seqs paired.qza --m-forward-barcodes-file Primers.tsv --m-forward-barcodes-column forward-barcode-seq --m-reverse-barcodes-file Primers.tsv --m-reverse-barcodes-column reverse-barcode-seq --o-per-sample-sequences demux-f-r.qza --o-untrimmed-sequences untrim-f-r.qza

qiime demux summarize --i-data demux-f-r.qza --o-visualization demux-f-r.qzv

qiime tools view demux-f-r.qzv

Am I doing something wrong?
Thanks!

[thermokarst]

Hi @Viktoria, can you please rerun the command, but include the --verbose flag this time? This will help us understand what cutadapt (the tool behind q2-cutadapt) did or didn't find in your multiplexed reads.

[Viktoria]

I reran the command with this flag. I am attaching screenshots.

[thermokarst]

Thanks @Viktoria - as you can see in your logs, cutadapt is failing to find the adapters you have specified. Please double-check that they are in the correct orientation and/or complement. You can learn more about cutadapt at Cutadapt — Cutadapt 3.5 documentation.