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export_filter.sh
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export_filter.sh
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#!/bin/bash
#SBATCH --job-name=TableExport_Filtering
#SBATCH --ntasks 1
#SBATCH --mem=48G
#SBATCH --output=stdout_tableexportFiltering.Log
#SBATCH --cpus-per-task 8
# Setting the working directory
cd /home/scarpettad
# Setting the path to the singularity container
sing_container=/home/scarpettad/amplicon_2023.9.sif
# Activate conda environment with biom
source activate /home/scarpettad/.conda/envs/metagen
# Setting metadata path
metadata=/home/scarpettad/metadata_secondrun.tsv
# Setting ASV table path
asv=/home/scarpettad/second_run/denoise_dada2/table-dada2.qza
# Setting the rep seqs path
rep_seqs=/home/scarpettad/second_run/denoise_dada2/rep-seqs-dada2.qza
# Setting classified seqs path
classified_seqs=/home/scarpettad/second_run/training_feature_classifier/classified-rep-seqs.qza
# Setting output dir
output_dir=/home/scarpettad/second_run/qiime2_taxonomy_output
# Export taxa bar plots without filterings:
singularity run $sing_container qiime taxa barplot \
--i-table $asv \
--i-taxonomy $classified_seqs \
--m-metadata-file $metadata \
--o-visualization $output_dir/taxa-barplots-classified-SILVA138-99.qzv
# Keep all sequences with classification at least at phylum level (other are left visible only in qiime2 taxa bar plot for QC purposes)
# Include only sequences classified at phylum level and remove chloroplast sequences from the table artifact
singularity run $sing_container qiime taxa filter-table \
--i-table $asv \
--i-taxonomy $classified_seqs \
--p-include p__ \
--p-exclude chloroplast \
--o-filtered-table $output_dir/filtered-table.qza
# Include only sequences classified at phylum level and remove chloroplast sequences from the rep-seqs artifact
singularity run $sing_container qiime taxa filter-seqs \
--i-sequences $rep_seqs \
--i-taxonomy $classified_seqs \
--p-include p__ \
--p-exclude chloroplast \
--o-filtered-sequences $output_dir/filtered-rep-seqs.qza
filtered_table=filtered-table.qza
filtered_sequences=filtered-rep-seqs.qza
# Export as .qzv to assess sequencing depth of beta diversity analysis
singularity run $sing_container qiime feature-table summarize \
--i-table $output_dir/$filtered_table \
--o-visualization $output_dir/filtered_table.qzv \
--m-sample-metadata-file $metadata
# Export filtered taxa bar plot:
singularity run $sing_container qiime taxa barplot \
--i-table $output_dir/$filtered_table \
--i-taxonomy $classified_seqs \
--m-metadata-file $metadata \
--o-visualization $output_dir/taxa-barplots-classified-SILVA138-99-filtered.qzv
##### Only filtered table and sequences are used for all the analysis
## Export taxonomic counts at all levels
# Export taxonomy as .tsv file
singularity run $sing_container qiime tools export \
--input-path $classified_seqs \
--output-path $output_dir/taxonomy
# Change header to taxonomy.tsv file
sed -i 's/Feature ID\tTaxon\tConfidence/#OTUID\ttaxonomy\tconfidence/' $output_dir/taxonomy/taxonomy.tsv
##### Export feature table at ASV level
##### Export filtered table in .biom format
singularity run $sing_container qiime tools export \
--input-path $output_dir/$filtered_table \
--output-path $output_dir/taxonomy
# Add taxonomy to FILTERED feature tables:
biom add-metadata \
-i $output_dir/taxonomy/feature-table.biom \
-o $output_dir/taxonomy/filtered-feature-table-with-taxonomy.biom \
--observation-metadata-fp $output_dir/taxonomy/taxonomy.tsv \
--sc-separated taxonomy
# Convert feature table with taxonomy (.biom to .tsv)
biom convert \
-i $output_dir/taxonomy/filtered-feature-table-with-taxonomy.biom \
-o $output_dir/taxonomy/filtered-feature-table-with-taxonomy.tsv \
--to-tsv \
--header-key taxonomy
# Delete first line of each feature table file
sed -i '1d' $output_dir/taxonomy/filtered-feature-table-with-taxonomy.tsv
##### Collapse feature-tables
# Collapse groups of features that have the same taxonomic assignment through the specified level. The frequencies of all features will be summed when they are collapsed.
for i in {1..7};
do singularity run $sing_container qiime taxa collapse \
--i-table $output_dir/$filtered_table \
--i-taxonomy $classified_seqs \
--p-level $i \
--o-collapsed-table $output_dir/taxonomy/table-level-$i.qza;
done
# Convert collapsed table (.qza to biom)
for i in {1..7};
do singularity run $sing_container qiime tools export \
--input-path $output_dir/taxonomy/table-level-$i.qza \
--output-path $output_dir/taxonomy/level-$i;
done
# Copy and change names to .biom tables adding the level of the collapse:
for i in {1..7};
do cp $output_dir/taxonomy/level-$i/feature-table.biom $output_dir/taxonomy/feature-table-$i.biom;
done
# Add taxonomy to feature tables
for i in {1..7};
do biom add-metadata \
-i $output_dir/taxonomy/feature-table-$i.biom \
-o $output_dir/taxonomy/feature-table-$i-with-taxonomy.biom \
--observation-metadata-fp $output_dir/taxonomy/taxonomy.tsv \
--sc-separated taxonomy;
done
# Convert feature table with taxonomy (.biom to .tsv)
for i in {1..7};
do biom convert \
-i $output_dir/taxonomy/feature-table-$i-with-taxonomy.biom \
-o $output_dir/taxonomy/feature-table-$i.tsv \
--to-tsv \
--header-key taxonomy;
done
# Delete first line of each feature table file
for i in {1..7};
do sed -i '1d' $output_dir/taxonomy/feature-table-$i.tsv;
done
# Remove taxonomy analysis intermediate files
rm $output_dir/taxonomy/*.biom
rm $output_dir/taxonomy/*.qza
# Deactivate conda environment
conda deactivate