Vsearch cluster-features-closed-reference error

I ran this trying to generate a closed reference file which I would then use to generate a BIOM file for analysis by PICRUSt and I received the following error

qiime vsearch cluster-features-closed-reference \
  --i-table table.qza \
  --i-sequences rep-seqs.qza \
  --i-reference-sequences gg_13_5_otu_97.qza \
  --p-perc-identity 0.97 \
  --o-clustered-table table-cr-97.qza \
  --o-unmatched-sequences unmatched.qza \
--o-clustered-sequences clustered-97.qza --verbose

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: vsearch --usearch_global /var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/tmp6ht_wxt6 --id 0.97 --db /var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/qiime2-archive-cnb2wt1r/9cd78e40-20be-4f7c-a495-b12510fd02ae/data/dna-sequences.fasta --uc /var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/tmpco0rzrrw --strand plus --qmask none --notmatched /var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/tmp0zppmc4e --threads 1

vsearch v2.7.0_macos_x86_64, 16.0GB RAM, 4 cores

Reading file /var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/qiime2-archive-cnb2wt1r/9cd78e40-20be-4f7c-a495-b12510fd02ae/data/dna-sequences.fasta 0%

Fatal error: illegal character '-' on line 2 in FASTA file
Plugin error from vsearch:

  Command '['vsearch', '--usearch_global', '/var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/tmp6ht_wxt6', '--id', '0.97', '--db', '/var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/qiime2-archive-cnb2wt1r/9cd78e40-20be-4f7c-a495-b12510fd02ae/data/dna-sequences.fasta', '--uc', '/var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/tmpco0rzrrw', '--strand', 'plus', '--qmask', 'none', '--notmatched', '/var/folders/7m/101qd31s36l0d0d1g6683cww0000gn/T/tmp0zppmc4e', '--threads', '1']' returned non-zero exit status 1

Good morning,

I think this is the important part of the error:

Looks like there is an issue with one of your fasta files! How did you import these into qiime artifacts? Are any of these aligned with gaps in them?


1 Like

So the reference sequences (97_otus.fasta) I used to generate the


was actually from the rep_set_aligned not the rep_set folder of the gg_13_5_otus. Using the 97_otus.fasta file from the rep_set folder gave me the appropriate outputs



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