Error: qiime vsearch cluster-features-open-reference command

When I run the following command:
qiime vsearch cluster-features-open-reference
–i-table table.qza
–i-sequences rep-seqs.qza
–i-reference-sequences unite_public_01.12.2017.qza
–p-perc-identity 0.85
–o-clustered-table table-or-85.qza\
–o-clustered-sequences rep-seqs-or-85.qza
–o-new-reference-sequences new-ref-seqs-or-85.qza\

I meet the problem:
Plugin error from vsearch:
Invalid character in sequence: b’g’.
Valid characters: [’-’, ‘N’, ‘K’, ‘Y’, ‘G’, ‘M’, ‘S’, ‘R’, ‘A’, ‘W’, ‘H’, ‘T’, ‘D’, ‘B’, ‘V’, ‘C’, ‘.’]
Note: Use lowercase if your sequence contains lowercase characters not in the sequence’s alphabet.

Then, I checked the raw sample data file (fasta) that I imported (qiime tools import --input-path seqs.fasta --output-path seqs.qza --type ‘SampleData[Sequences]’), and also checked the file ‘rep-seqs.qza’. However, I cannot find the lowercase letter ‘g’ in my raw sequences and representative sequences.

How can I fixed? Thanks if anybody here can help!

Hi @Naili, Have you performed the same check for lowercase characters on your reference sequences? It’s possible that lowercase characters is in there would result in this error.

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Thank you for the suggestion! I have checked it. Exactly, the error is caused by reference sequences.

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