S5 Supplementary Information

Be positive: customised reference databases and new, local barcodes balance false taxonomic assignments in metabarcoding studies

List of commands to produce customized reference databases and for taxonomic assignments of metabarcoding data.

Tools used in this workflow

• COInr DB https://zenodo.org/record/6555985, (Meglécz, 2022a, 2022b)
• mkCOInr v-0.2.0 https://github.com/meglecz/mkCOInr, (Meglécz, 2022b, 2022c)
• VTAM v-0.2.0 https://github.com/aitgon/vtam (González et al., 2020)
• RDP classifier v-2.13 https://sourceforge.net/p/rdp-classifier/news/2020/07/rdp-classifier-213-july-2020-release-note/, (Wang et al., 2007)
• QIIME2 v-Core-2022.2 https://docs.qiime2.org/2022.8/install/, (Bolyen et al., 2019)

1. Select sequences from COInr

1.1 Download COInr DB

Downloaded from www.zenodo.org/record/6555985. This DB is ready to use as it is for formatting and for selecting a desired target region. It comes with an associated taxonomy .tsv file.

mkdir -p ~/DB_comp
cd ~/DB_comp
wget https://zenodo.org/record/6555985/files/COInr_2022_05_06.tar.gz
tar -zxvf COInr_2022_05_06.tar.gz
mv COInr_2022_05_06 COInr
rm COInr_2022_05_06.tar.gz

1.2 COInr-WO-Insecta

Remove insect sequences from COInr

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perl ~/mkCOInr/scripts/select_taxa.pl \
  -taxon_list metafiles/taxon_list_insecta.txt \
  -tsv COInr/COInr.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outdir COInr_WO_Insecta/ \
  -out COInr_WO_Insecta.tsv \
  -negative_list 1

metafiles/taxon_list_insecta.txt:

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taxon_name
Insecta

1.3 COInr-Med

Derived from COInr-WO-Insecta and refined for Mediterranean marine Families gathered from OBIS*

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perl ~/mkCOInr/scripts/select_taxa.pl \
  -taxon_list metafiles/Data_S4.tsv \
  -tsv COInr_WO_Insecta/COInr_WO_Insecta.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outdir COInr_Med/ \
  -out COInr_Med.tsv \
  -negative_list 0

Data_S4.tsv is the list of taxonomic families present in the Mediterranean Sea.

1.4 COInr-Med+

Add new barcodes to COInr-Med

1.4.1 Suggest one or more lineages for each taxon name based on the existing lineages in taxonomy.tsv

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  perl ~/mkCOInr/scripts/format_custom.pl \
  -custom metafiles/Data_S2_barcodes.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outdir COInr_Med_plus/format_custom

Data_S2_barcodes.tsv is a tab separated file with seqID, taxon, sequence as columns. It can be created from Data_S2.tsv by selecting the appropriate columns.

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seqID   taxon      sequence
Seq1 Orbinia sertulata      ATCAGTAGATATAGCAATC...
Seq10    Achelia langi      TTTATCATCGAGATTGGC...

The output is a lineage file COInr_Med_plus/format_custom/custom_lineages.tsv and a sequence file COInr_Med_plus/format_custom/custom_sequences.tsv.

Revise the output lineage file to complete lineages if taxon name is new to taxonomy.tsv and choose between homonyms if necessary.

custom_lineages_verified.tsv

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phylum  class   order   family  subfamily       genus   species seqIDs
Cnidaria    Anthozoa    Pennatulacea    Funiculinidae       Funiculina  Funiculina quadrangularis   Seq103;Seq130
...

1.4.2 Find taxID for each taxon in the lineage file

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perl ~/mkCOInr/scripts/add_taxids.pl \
  -lineages metafiles/custom_lineages_verified.tsv \
  -sequences COInr_Med_plus/format_custom/custom_sequences.tsv \
  -outdir COInr_Med_plus/add_taxids \
  -taxonomy COInr/taxonomy.tsv

This command will update the taxonomy.tsv file by adding new taxIDs. Remember to use the generated COInr_Med_plus/add_taxids/taxonomy_updated.tsv file for further taxonomic assignation steps. It will also produce COInr_Med_plus/add_taxids/sequences_with_taxIDs.tsv used in the next step.

1.4.3 Dereplicate custom sequences

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perl ~/mkCOInr/scripts/dereplicate.pl \
  -tsv COInr_Med_plus/add_taxids/sequences_with_taxIDs.tsv \
  -outdir COInr_Med_plus/dereplicate \
  -out custom_dereplicated_sequences.tsv

1.4.4 Pool and dereplicate COInr_Med DB + custom sequences

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perl ~/mkCOInr/scripts/pool_and_dereplicate.pl \
  -tsv1 COInr_Med/COInr_Med.tsv \
  -tsv2 COInr_Med_plus/dereplicate/custom_dereplicated_sequences.tsv \
  -outdir COInr_Med_plus \
  -out COInr_Med_plus.tsv

Move the updated taxonomy file to the same folder as the COInr_Med_plus.tsv.

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mv COInr_Med_plus/add_taxids/taxonomy_updated.tsv COInr_Med_plus

2. Select the Leray region for each DB

Select sequences that cover at least 80% of the region amplified by metabarcoding primer pairs and trim sequences to this region.

2.1 COInr

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perl ~/mkCOInr/scripts/select_region.pl \
  -tsv COInr/COInr.tsv \
  -outdir leray/COInr \
  -e_pcr 1 \
  -fw GGNTGAACNGTNTAYCCNCC \
  -rv TAWACTTCDGGRTGNCCRAARAAYCA \
  -trim_error 0.3 \
  -min_amplicon_length 280 \
  -max_amplicon_length 345 \
  -min_overlap 20 \
  -tcov 0.8 \
  -identity 0.7

2.2 COInr-WO-Insecta

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perl ~/mkCOInr/scripts/select_region.pl \
  -tsv COInr_WO_Insecta/COInr_WO_Insecta.tsv \
  -outdir leray/COInr_WO_Insecta \
  -e_pcr 1 \
  -fw GGNTGAACNGTNTAYCCNCC \
  -rv TAWACTTCDGGRTGNCCRAARAAYCA \
  -trim_error 0.3 \
  -min_amplicon_length 280 \
  -max_amplicon_length 345 \
  -min_overlap 20 \
  -tcov 0.8 \
  -identity 0.7

2.3 COInr-Med

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perl ~/mkCOInr/scripts/select_region.pl \
  -tsv COInr_Med/COInr_Med.tsv \
  -outdir leray/COInr_Med \
  -e_pcr 1 \
  -fw GGNTGAACNGTNTAYCCNCC \
  -rv TAWACTTCDGGRTGNCCRAARAAYCA \
  -trim_error 0.3 \
  -min_amplicon_length 280 \
  -max_amplicon_length 345 \
  -min_overlap 20 \
  -tcov 0.8 \
  -identity 0.7

2.4 COInr-Med+

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perl ~/mkCOInr/scripts/select_region.pl \
  -tsv COInr_Med_plus/COInr_Med_plus.tsv \
  -outdir leray/COInr_Med_plus \
  -e_pcr 1 \
  -fw GGNTGAACNGTNTAYCCNCC \
  -rv TAWACTTCDGGRTGNCCRAARAAYCA \
  -trim_error 0.3 \
  -min_amplicon_length 280 \
  -max_amplicon_length 345 \
  -min_overlap 20 \
  -tcov 0.8 \
  -identity 0.7

3. Format the four databases for each taxassing tools

3.1 VTAM format

3.1.1 COInr

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt vtam \
  -outdir leray/COInr/vtam \
  -out COInr_vtam

3.1.2 COInr-WO-Insecta

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_WO_Insecta/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt vtam \
  -outdir leray/COInr_WO_Insecta/vtam \
  -out COInr_WO_Insecta_vtam

3.1.3 COInr-Med

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_Med/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt vtam \
  -outdir leray/COInr_Med/vtam \
  -out COInr_Med_vtam

3.1.4 COInr-Med+

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_Med_plus/trimmed.tsv \
  -taxonomy COInr_Med_plus/taxonomy_updated.tsv \
  -outfmt vtam \
  -outdir leray/COInr_Med_plus/vtam \
  -out COInr_Med_plus_vtam

3.2 RDP format

3.2.1 COInr

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt rdp \
  -outdir leray/COInr/rdp \
  -out COInr_rdp

3.2.2 COInr-WO-Insecta

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_WO_Insecta/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt rdp \
  -outdir leray/COInr_WO_Insecta/rdp \
  -out COInr_WO_Insecta_rdp

3.2.3 COInr-Med

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_Med/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt rdp \
  -outdir leray/COInr_Med/rdp \
  -out COInr_Med_rdp

3.2.4 COInr-Med+

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_Med_plus/trimmed.tsv \
  -taxonomy COInr_Med_plus/taxonomy_updated.tsv \
  -outfmt rdp \
  -outdir leray/COInr_Med_plus/rdp \
  -out COInr_Med_plus_rdp

3.3 QIIME format

3.3.1 COInr

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt qiime \
  -outdir leray/COInr/qiime \
  -out COInr_qiime

3.3.2 COInr-WO-Insecta

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_WO_Insecta/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt qiime \
  -outdir leray/COInr_WO_Insecta/qiime \
  -out COInr_WO_Insecta_qiime

3.3.3 COInr-Med

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_Med/trimmed.tsv \
  -taxonomy COInr/taxonomy.tsv \
  -outfmt qiime \
  -outdir leray/COInr_Med/qiime \
  -out COInr_Med_qiime

3.3.4 COInr-Med+

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perl ~/mkCOInr/scripts/format_db.pl \
  -tsv leray/COInr_Med_plus/trimmed.tsv \
  -taxonomy COInr_Med_plus/taxonomy_updated.tsv \
  -outfmt qiime \
  -outdir leray/COInr_Med_plus/qiime \
  -out COInr_Med_plus_qiime

4. Taxonomic assignment

4.1 VTAM taxassign

Create output directory

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mkdir -p taxassign/vtam

4.1.1 COInr

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vtam taxassign \
  --mode reset \
  --db metafiles/db.sqlite \
  --asvtable metafiles/Data_S3.tsv \
  --output taxassign/vtam/COInr_vtam_taxassign.tsv \
  --taxonomy leray/COInr/vtam/COInr_vtam_taxonomy.tsv \
  --blastdbdir leray/COInr/vtam/ \
  --blastdbname COInr_vtam \
  -v

4.1.2 COInr-WO-Insecta

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vtam taxassign \
  --mode reset \
  --db metafiles/db.sqlite \
  --asvtable metafiles/Data_S3.tsv \
  --output taxassign/vtam/COInr_WO_Insecta_vtam_taxassign.tsv \
  --taxonomy leray/COInr_WO_Insecta/vtam/COInr_WO_Insecta_vtam_taxonomy.tsv \
  --blastdbdir leray/COInr_WO_Insecta/vtam/ \
  --blastdbname COInr_WO_Insecta_vtam \
  -v

4.1.3 COInr-Med

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vtam taxassign \
  --mode reset \
  --db metafiles/db.sqlite \
  --asvtable metafiles/Data_S3.tsv \
  --output taxassign/vtam/COInr_Med_vtam_taxassign.tsv \
  --taxonomy leray/COInr_Med/vtam/COInr_Med_vtam_taxonomy.tsv \
  --blastdbdir leray/COInr_Med/vtam/ \
  --blastdbname COInr_Med_vtam \
  -v

4.1.4 COInr-Med+

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vtam taxassign \
  --mode reset \
  --db metafiles/db.sqlite \
  --asvtable metafiles/Data_S3.tsv \
  --output taxassign/vtam/COInr_Med_plus_vtam_taxassign.tsv \
  --taxonomy leray/COInr_Med_plus/vtam/COInr_Med_plus_vtam_taxonomy.tsv \
  --blastdbdir leray/COInr_Med_plus/vtam/ \
  --blastdbname COInr_Med_plus_vtam \
  -v

4.2 RDP classifier

4.2.1 RDP training

The "Xmx216g" command has to be adjusted according to your available RAM (e.g., 216 = 216GB). Do not use all the available RAM of your machine, it will freeze.

Create output directories

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mkdir -p leray/COInr/rdp/trained
mkdir -p leray/COInr_WO_Insecta/rdp/trained
mkdir -p leray/COInr_Med/rdp/trained
mkdir -p leray/COInr_Med_plus/rdp/trained

COInr

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  train \
  -o leray/COInr/rdp/trained/ \
  -s leray/COInr/rdp/COInr_rdp_trainseq.fasta \
  -t leray/COInr/rdp/COInr_rdp_taxon.txt

COInr-WO-Insecta

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  train \
  -o leray/COInr_WO_Insecta/rdp/trained/ \
  -s leray/COInr_WO_Insecta/rdp/COInr_WO_Insecta_rdp_trainseq.fasta \
  -t leray/COInr_WO_Insecta/rdp/COInr_WO_Insecta_rdp_taxon.txt

COInr-Med

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  train \
  -o leray/COInr_Med/rdp/trained/ \
  -s leray/COInr_Med/rdp/COInr_Med_rdp_trainseq.fasta \
  -t leray/COInr_Med/rdp/COInr_Med_rdp_taxon.txt

COInr-Med+

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  train \
  -o leray/COInr_Med_plus/rdp/trained/ \
  -s leray/COInr_Med_plus/rdp/COInr_Med_plus_rdp_trainseq.fasta \
  -t leray/COInr_Med_plus/rdp/COInr_Med_plus_rdp_taxon.txt

4.2.2 Taxonomic assigment with RDP classifier

Create output directory

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mkdir -p taxassign/rdp

COInr

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  classify \
  -t leray/COInr/rdp/trained/rRNAClassifier.properties \
  -o taxassign/rdp/COInr_rdp_taxassign.tsv \
  metafiles/Data_S3.fasta

COInr-WO-Insecta

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  classify \
  -t leray/COInr_WO_Insecta/rdp/trained/rRNAClassifier.properties \
  -o taxassign/rdp/COInr_WO_Insecta_rdp_taxassign.tsv \
  metafiles/Data_S3.fasta

COInr-Med

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  classify \
  -t leray/COInr_Med/rdp/trained/rRNAClassifier.properties \
  -o taxassign/rdp/COInr_Med_rdp_taxassign.tsv \
  metafiles/Data_S3.fasta

COInr-Med+

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java \
  -Xmx216g \
  -jar rdp_classifier_2.13/rdp_classifier_2.13/dist/classifier.jar \
  classify \
  -t leray/COInr_Med_plus/rdp/trained/rRNAClassifier.properties \
  -o taxassign/rdp/COInr_Med_plus_rdp_taxassign.tsv \
  metafiles/Data_S3.fasta

4.3 Taxonomic assignment with QIIME2

4.3.1 Import database sequences and taxonomy to QIIME2

COInr

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qiime tools import \
  --type 'FeatureData[Sequence]' \
  --input-path leray/COInr/qiime/COInr_qiime_trainseq.fasta \
  --output-path leray/COInr/qiime/COInr_sequences.qza

qiime tools import \
  --type 'FeatureData[Taxonomy]' \
  --input-format HeaderlessTSVTaxonomyFormat \
  --input-path leray/COInr/qiime/COInr_qiime_taxon.txt \
  --output-path leray/COInr/qiime/COInr_taxonomy.qza

COInr-WO-Insecta

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qiime tools import \
  --type 'FeatureData[Sequence]' \
  --input-path leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_qiime_trainseq.fasta \
  --output-path leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_sequences.qza

qiime tools import \
  --type 'FeatureData[Taxonomy]' \
  --input-format HeaderlessTSVTaxonomyFormat \
  --input-path leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_qiime_taxon.txt \
  --output-path leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_taxonomy.qza

COInr-Med

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qiime tools import \
  --type 'FeatureData[Sequence]' \
  --input-path leray/COInr_Med/qiime/COInr_Med_qiime_trainseq.fasta \
  --output-path leray/COInr_Med/qiime/COInr_Med_sequences.qza

qiime tools import \
  --type 'FeatureData[Taxonomy]' \
  --input-format HeaderlessTSVTaxonomyFormat \
  --input-path leray/COInr_Med/qiime/COInr_Med_qiime_taxon.txt \
  --output-path leray/COInr_Med/qiime/COInr_Med_taxonomy.qza

COInr-Med+

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qiime tools import \
  --type 'FeatureData[Sequence]' \
  --input-path leray/COInr_Med_plus/qiime/COInr_Med_plus_qiime_trainseq.fasta \
  --output-path leray/COInr_Med_plus/qiime/COInr_Med_plus_sequences.qza

qiime tools import \
  --type 'FeatureData[Taxonomy]' \
  --input-format HeaderlessTSVTaxonomyFormat \
  --input-path leray/COInr_Med_plus/qiime/COInr_Med_plus_qiime_taxon.txt \
  --output-path leray/COInr_Med_plus/qiime/COInr_Med_plus_taxonomy.qza

4.3.2 Import the test ASV dataset to QIIME artifact

Sequences should be in CAPITAL letters

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qiime tools import \
--type 'FeatureData[Sequence]' \
--input-path metafiles/Data_S3.fasta \
--output-path metafiles/Data_S3.qza

4.3.3 Train Qiime classifier

COInr

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qiime feature-classifier fit-classifier-naive-bayes \
  --i-reference-reads leray/COInr/qiime/COInr_sequences.qza \
  --i-reference-taxonomy leray/COInr/qiime/COInr_taxonomy.qza \
  --o-classifier leray/COInr/qiime/COInr_trained.qza

COInr-WO-Insecta

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qiime feature-classifier fit-classifier-naive-bayes \
  --i-reference-reads leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_sequences.qza \
  --i-reference-taxonomy leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_taxonomy.qza \
  --o-classifier leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_trained.qza

COInr-Med

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qiime feature-classifier fit-classifier-naive-bayes \
  --i-reference-reads leray/COInr_Med/qiime/COInr_Med_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med/qiime/COInr_Med_taxonomy.qza \
  --o-classifier leray/COInr_Med/qiime/COInr_Med_trained.qza

COInr-Med+

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qiime feature-classifier fit-classifier-naive-bayes \
  --i-reference-reads leray/COInr_Med_plus/qiime/COInr_Med_plus_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med_plus/qiime/COInr_Med_plus_taxonomy.qza \
  --o-classifier leray/COInr_Med_plus/qiime/COInr_Med_plus_trained.qza

4.3.4 Classify (taxassign) with QIIME2 using SKLEARN algorithm

Create output directory

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mkdir -p taxassign/qiime_sklearn

COInr

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qiime feature-classifier classify-sklearn \
  --i-classifier leray/COInr/qiime/COInr_trained.qza \
  --i-reads metafiles/Data_S3.qza \
  --o-classification taxassign/qiime_sklearn/COInr_qiime_sklearn_taxassign.qza

COInr-WO-Insecta

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qiime feature-classifier classify-sklearn \
  --i-classifier leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_trained.qza \
  --i-reads metafiles/Data_S3.qza \
  --o-classification taxassign/qiime_sklearn/COInr_WO_Insecta_qiime_sklearn_taxassign.qza

COInr-Med

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qiime feature-classifier classify-sklearn \
  --i-classifier leray/COInr_Med/qiime/COInr_Med_trained.qza \
  --i-reads metafiles/Data_S3.qza \
  --o-classification taxassign/qiime_sklearn/COInr_Med_qiime_sklearn_taxassign.qza

COInr-Med+

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qiime feature-classifier classify-sklearn \
  --i-classifier leray/COInr_Med_plus/qiime/COInr_Med_plus_trained.qza \
  --i-reads metafiles/Data_S3.qza \
  --o-classification taxassign/qiime_sklearn/COInr_Med_plus_qiime_sklearn_taxassign.qza

4.3.5 Classify (taxassign) with QIIME2 using BLAST algorithm

Use three different percentage of identity: 0.97, 0.9, 0.8

Create output directory

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mkdir -p taxassign/qiime_blast

COInr

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qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr/qiime/COInr_sequences.qza \
  --i-reference-taxonomy leray/COInr/qiime/COInr_taxonomy.qza \
  --p-perc-identity 0.97 \
  --o-classification taxassign/qiime_blast/COInr_qiime_blast_97_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr/qiime/COInr_sequences.qza \
  --i-reference-taxonomy leray/COInr/qiime/COInr_taxonomy.qza \
  --p-perc-identity 0.90 \
  --o-classification taxassign/qiime_blast/COInr_qiime_blast_90_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr/qiime/COInr_sequences.qza \
  --i-reference-taxonomy leray/COInr/qiime/COInr_taxonomy.qza \
  --p-perc-identity 0.80 \
  --o-classification taxassign/qiime_blast/COInr_qiime_blast_80_taxassign.qza \
  --verbose

COInr-WO-Insecta

xxxxxxxxxx
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_sequences.qza \
  --i-reference-taxonomy leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_taxonomy.qza \
  --p-perc-identity 0.97 \
  --o-classification taxassign/qiime_blast/COInr_WO_Insecta_qiime_blast_97_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_sequences.qza \
  --i-reference-taxonomy leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_taxonomy.qza \
  --p-perc-identity 0.90 \
  --o-classification taxassign/qiime_blast/COInr_WO_Insecta_qiime_blast_90_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_sequences.qza \
  --i-reference-taxonomy leray/COInr_WO_Insecta/qiime/COInr_WO_Insecta_taxonomy.qza \
  --p-perc-identity 0.80 \
  --o-classification taxassign/qiime_blast/COInr_WO_Insecta_qiime_blast_80_taxassign.qza \
  --verbose

COInr-Med

xxxxxxxxxx
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_Med/qiime/COInr_Med_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med/qiime/COInr_Med_taxonomy.qza \
  --p-perc-identity 0.97 \
  --o-classification taxassign/qiime_blast/COInr_Med_qiime_blast_97_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_Med/qiime/COInr_Med_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med/qiime/COInr_Med_taxonomy.qza \
  --p-perc-identity 0.90 \
  --o-classification taxassign/qiime_blast/COInr_Med_qiime_blast_90_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_Med/qiime/COInr_Med_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med/qiime/COInr_Med_taxonomy.qza \
  --p-perc-identity 0.80 \
  --o-classification taxassign/qiime_blast/COInr_Med_qiime_blast_80_taxassign.qza \
  --verbose

COInr-Med+

xxxxxxxxxx
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_Med_plus/qiime/COInr_Med_plus_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med_plus/qiime/COInr_Med_plus_taxonomy.qza \
  --p-perc-identity 0.97 \
  --o-classification taxassign/qiime_blast/COInr_Med_plus_qiime_blast_97_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_Med_plus/qiime/COInr_Med_plus_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med_plus/qiime/COInr_Med_plus_taxonomy.qza \
  --p-perc-identity 0.90 \
  --o-classification taxassign/qiime_blast/COInr_Med_plus_qiime_blast_90_taxassign.qza \
  --verbose
  
qiime feature-classifier classify-consensus-blast \
  --i-metafiles/Data_S3.qza \
  --i-reference-reads leray/COInr_Med_plus/qiime/COInr_Med_plus_sequences.qza \
  --i-reference-taxonomy leray/COInr_Med_plus/qiime/COInr_Med_plus_taxonomy.qza \
  --p-perc-identity 0.80 \
  --o-classification taxassign/qiime_blast/COInr_Med_plus_qiime_blast_80_taxassign.qza \
  --verbose

5. References

Bolyen E, et al. 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature Biotechnology 37:852–857. DOI: 10.1038/s41587-019-0209-9.Microbiome, 6, 90.

González,A. et al. (2020) VTAM: A robust pipeline for validating metabarcoding data using internal controls. bioRxiv, 2020.11.06.371187.

Meglécz,E. (2022a) COInr a comprehensive, non-redundant COI database from NCBI-nt and BOLD. DOI: 10.5281/zenodo.6555985.

Meglécz,E. (2022b) COInr and mkCOInr: Building and customizing a non-redundant barcoding reference database from BOLD and NCBI using a lightweight pipeline. BioRxiv:2022.05.18.492423.

Meglécz,E. (2022c) meglecz/mkCOInr: mkCOInr-v.0.2.0. DOI: 10.5281/zenodo.6961340

Wang,Q. et al. (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol., 73, 5261–5267.