Get_silva-138&filter.md

Workflow

Downloading SILVA data

qiime rescript get-silva-data --p-version '138' --p-target 'SSURef_NR99' --p-include-species-labels --o-silva-sequences silva-138-ssu-nr99-seqs.qza --o-silva-taxonomy silva-138-ssu-nr99-tax.qza

Taxonomy file has 7 levels as required and without gaps (all levels are labelled). Not performing the rank propagation step.

Removing low quality sequences

qiime rescript cull-seqs --i-sequences silva-138-ssu-nr99-seqs.qza --o-clean-sequences silva-138-ssu-nr99-seqs-cleaned.qza

Remove Eukaryotic taxa

qiime taxa filter-seqs --i-sequences silva-138-ssu-nr99-seqs-cleaned.qza --i-taxonomy silva-138-ssu-nr99-tax.qza --p-exclude 'd__Eukaryota' --p-mode 'contains' --o-filtered-sequences silva138_noEuk_seqs.qza

Filter by length

Archaea, Bacteria & Eukaryota (900, 1200, 1400 bases) - Excluding Eukaryota as it has already been removed.

qiime rescript filter-seqs-length-by-taxon --i-sequences silva138_noEuk_seqs.qza --i-taxonomy silva-138-ssu-nr99-tax.qza --p-labels Archaea Bacteria --p-min-lens 900 1200 --o-filtered-seqs silva138_noEuk_AB_seqs.qza --o-discarded-seqs silva138_Euk_seqs_discard.qza

This step did not discard any sequences, as those sequences were filtered in the previous step itself

Dereplicating

Default mode - uniq; Default rank-handles - silva

qiime rescript dereplicate --i-sequences silva138_noEuk_AB_seqs.qza --i-taxa silva-138-ssu-nr99-tax.qza --p-threads 12 --o-dereplicated-sequences silva138_noEuk_AB_seqs_uniq.qza --o-dereplicated-taxa silva138_noEuk_AB_tax_uniq.qza

Dereplicated Sequences: silva138_noEuk_AB_seqs_uniq.qza
Dereplicated Taxa: silva138_noEuk_AB_tax_uniq.qza

Classifying & evaluating with RESCRIPt

Generating a full length classifier

This step was performed with qiime_2023.5 on a Fedora 39 server

qiime rescript evaluate-fit-classifier --i-sequences silva138_noEuk_AB_seqs_uniq.qza --i-taxonomy silva138_noEuk_AB_tax_uniq.qza --o-classifier silva138_noEuk_AB_classifier.qza --o-observed-taxonomy silva138_noEuk_AB_predicted_taxonomy.qza --o-evaluation silva138_noEuk_AB_classifier_eval.qzv --p-n-jobs 10

Classifier: silva138_noEuk_AB_classifier.qza
Predicted Taxonomy: silva138_noEuk_AB_predicted_taxonomy.qza
Evaluation: silva138_noEuk_AB_classifier_eval.qzv

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• Workflow for V1-V2 classifier
• Workflow for V3 classifier
• Workflow for V3-V4 classifier
• Workflow for V4 classifier