Pyrosequence work in Qiime2

Hello, experts.
I am working with the pyrosequence dataset in Qiime2. I followed this amazing tutorial (analyzing 454 data in QIIME 2), and while applying it to my data, I encountered a problem with cutadapt.

qiime tools import
--type MultiplexedSingleEndBarcodeInSequence
--input-format MultiplexedFastaQualDirFmt
--input-path practice
--output-path practice_seqs.qza
(worked)

qiime cutadapt demux-single
--i-seqs practice_seqs.qza
--m-barcodes-file practice_Map.txt
--m-barcodes-column BarcodeSequence
--o-per-sample-sequences practice_demux.qza
--o-untrimmed-sequences unassigned.qza

Plugin error from cutadapt:

Command '['cutadapt', '--front', 'file:/var/folders/bd/kdj7l_yx1zvf2hfp96yc8s4w0000gn/T/tmp43w4bmv1', '--error-rate', '0.1', '--minimum-length', '1', '-o', '/var/folders/bd/kdj7l_yx1zvf2hfp96yc8s4w0000gn/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-5lsy40oc/{name}.1.fastq.gz', '--untrimmed-output', '/var/folders/bd/kdj7l_yx1zvf2hfp96yc8s4w0000gn/T/q2-MultiplexedSingleEndBarcodeInSequenceDirFmt-kt97c3bl/forward.fastq.gz', '/var/folders/bd/kdj7l_yx1zvf2hfp96yc8s4w0000gn/T/qiime2/GUAN_LAB/data/f2b32c33-b1c5-4ec6-abb6-119b2a720ad2/data/forward.fastq.gz', '-j', '1']' returned non-zero exit status 2.

Debug info has been saved to /var/folders/bd/kdj7l_yx1zvf2hfp96yc8s4w0000gn/T/qiime2-q2cli-err-roddkjrp.log

When I see the log files, "ERROR: Character 'P' in adapter sequence 'MPS077743' is not a valid IUPAC code. Use only characters 'ABCDGHIKMNRSTUVWXY'."

It seems that the barcode might be the problem. How can I address this issue? Any ideas would be appreciated.

practice_Map.txt

#SampleID BarcodeSequence LinkerPrimerSequence Treatment
1017.1.23 MPS077743 GAGTTTGATCMTGGCTCAG CON
1023.1.23 MPS077755 GAGTTTGATCMTGGCTCAG CON

Many thanks

1 Like

Hello @You_y_Choi,

You are correct!

These look like barcode names.
The column BarcodeSequence should be a sequence, like ATCGTGCTGCTA.