#!/bin/bash # Given metadata file path is /mnt/datasets/project_2/zoo/zoo_metadata.txt # Changes to zoo_metadata_split.txt and zoo_metadata_combined.txt # Removed “#” from “#SampleID” header # Replaced “/” with “_” in metadata headers # Additional changes only to zoo_metadata_combined.txt # Combined “LeastConcern” and “NearThreatened” into “LCNT” under “ConservationStatusHMC” header # Combined “Vulnerable”, “Endangered”, and “CriticallyEndangered” into “VECE” under “ConservationStatusHMC” header ### Processing reads # Import and demultiplex data echo Importing and demultiplexing data... qiime tools import --type 'SampleData[SequencesWithQuality]' \ --input-path /mnt/datasets/project_2/zoo/zoo_manifest.txt \ --input-format SingleEndFastqManifestPhred33V2 \ --output-path demux.qza # Create visualization of demultiplexed samples qiime demux summarize \ --i-data demux.qza \ --o-visualization demux.qzv # Trim, denoise, and determine ASVs with DADA2 echo Trimming, denoising, and determining ASVs... qiime dada2 denoise-single \ --i-demultiplexed-seqs demux.qza \ --p-trim-left 0 \ --p-trunc-len 234 \ --o-representative-sequences rep-seqs.qza \ --o-table table.qza \ --o-denoising-stats stats.qza # Visualize DADA2 output statistics qiime metadata tabulate \ --m-input-file stats.qza \ --o-visualization stats.qzv # Visualize ASV output statistics qiime feature-table summarize \ --i-table table.qza \ --o-visualization table.qzv \ --m-sample-metadata-file zoo_metadata_split.txt qiime feature-table tabulate-seqs \ --i-data rep-seqs.qza \ --o-visualization rep-seqs.qzv # Perform taxonomic analysis using the 16S SILVA reference database echo Assigning taxonomy... qiime feature-classifier classify-sklearn \ --i-classifier /mnt/datasets/classifiers/silva-138-99-515-806-nb-classifier.qza \ --i-reads rep-seqs.qza \ --o-classification taxonomy.qza qiime metadata tabulate \ --m-input-file taxonomy.qza \ --o-visualization taxonomy.qzv # Generate taxonomy barplots qiime taxa barplot \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --m-metadata-file zoo_metadata_split.txt \ --o-visualization taxa-bar-plots.qzv # Remove mitochondria and chloroplast sequences echo Filtering out mitochondria and chloroplast sequences... qiime taxa filter-table \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --p-exclude mitochondria,chloroplast \ --o-filtered-table table-no-mitochondria-no-chloroplast.qza # Generate a tree for phylogenetic diversity analyses echo Generating a phylogenetic tree... qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences rep-seqs.qza \ --o-alignment aligned-rep-seqs.qza \ --o-masked-alignment masked-aligned-rep-seqs.qza \ --o-tree unrooted-tree.qza \ --o-rooted-tree rooted-tree.qza ### Overall dataset analysis of conservation status ### Subset by None # Visualize alpha-rarefaction for overall dataset qiime diversity alpha-rarefaction \ --i-table table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 200000 \ --m-metadata-file zoo_metadata_split.txt \ --o-visualization overall-alpha-rarefaction.qzv # Calculate beta-diversity metrics for overall dataset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table-no-mitochondria-no-chloroplast.qza \ --p-sampling-depth 83570 \ --m-metadata-file zoo_metadata_split.txt \ --output-dir overall-core-metrics-results # Calculate beta-group-significance for conservation status for overall dataset qiime diversity beta-group-significance \ --i-distance-matrix overall-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_split.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization overall-core-metrics-results/overall-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix overall-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_split.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization overall-core-metrics-results/overall-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix overall-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_split.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization overall-core-metrics-results/overall-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix overall-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_split.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization overall-core-metrics-results/overall-weighted-unifrac-conservation-significance.qzv \ --p-pairwise ### Diet type subset analysis of conservation status ### Subset by “OchHMC” into C (Carnivores), H (Herbivores), or O (Omnivores) mkdir diet_type_filtered_tables ## Filter for carnivores qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[OchHMC]='C'" \ --o-filtered-table diet_type_filtered_tables/carn-filtered-table.qza # Visualize ASV output statistics for carnivore subset qiime feature-table summarize \ --i-table diet_type_filtered_tables/carn-filtered-table.qza \ --o-visualization diet_type_filtered_tables/carn-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Visualize alpha-rarefaction for carnivore subset qiime diversity alpha-rarefaction \ --i-table diet_type_filtered_tables/carn-filtered-table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 150000 \ --m-metadata-file zoo_metadata_combined.txt \ --o-visualization diet_type_filtered_tables/carn-alpha-rarefaction.qzv # Calculate beta-diversity metrics for carnivore subset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table diet_type_filtered_tables/carn-filtered-table.qza \ --p-sampling-depth 80508 \ --m-metadata-file zoo_metadata_combined.txt \ --output-dir diet_type_filtered_tables/carn-core-metrics-results # Calculate beta-group-significance for conservation status for carnivore subset qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/carn-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/carn-core-metrics-results/carn-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/carn-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/carn-core-metrics-results/carn-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/carn-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/carn-core-metrics-results/carn-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/carn-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/carn-core-metrics-results/carn-weighted-unifrac-conservation-significance.qzv \ --p-pairwise ## Filter for herbivores qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[OchHMC]='H'" \ --o-filtered-table diet_type_filtered_tables/herb-filtered-table.qza # Visualize ASV output statistics for herbivore subset qiime feature-table summarize \ --i-table diet_type_filtered_tables/herb-filtered-table.qza \ --o-visualization diet_type_filtered_tables/herb-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Visualize alpha-rarefaction for herbivore subset qiime diversity alpha-rarefaction \ --i-table diet_type_filtered_tables/herb-filtered-table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 200000 \ --m-metadata-file zoo_metadata_combined.txt \ --o-visualization diet_type_filtered_tables/herb-alpha-rarefaction.qzv # Calculate beta-diversity metrics for herbivore subset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table diet_type_filtered_tables/herb-filtered-table.qza \ --p-sampling-depth 80508 \ --m-metadata-file zoo_metadata_combined.txt \ --output-dir diet_type_filtered_tables/herb-core-metrics-results # Calculate beta-group-significance for conservation status for herbivore subset qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/herb-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/herb-core-metrics-results/herb-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/herb-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/herb-core-metrics-results/herb-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/herb-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/herb-core-metrics-results/herb-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_type_filtered_tables/herb-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_type_filtered_tables/herb-core-metrics-results/herb-weighted-unifrac-conservation-significance.qzv \ --p-pairwise ## Filter for omnivores qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[OchHMC]='O'" \ --o-filtered-table diet_type_filtered_tables/omni-filtered-table.qza # Visualize ASV output statistics for omnivore subset qiime feature-table summarize \ --i-table diet_type_filtered_tables/omni-filtered-table.qza \ --o-visualization diet_type_filtered_tables/omni-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Omnivore subset discarded because of insufficient samples ### Diet breadth subset analysis of conservation status ### Subset by “NumberDietCategories” into 1 (Single Diet Category) or 2-5 (Multiple Diet Categories) mkdir diet_breadth_filtered_tables ## Filter for single diet category samples qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[NumberDietCategories]='1'" \ --o-filtered-table diet_breadth_filtered_tables/single-cat-filtered-table.qza # Visualize ASV output statistics for single diet category subset qiime feature-table summarize \ --i-table diet_breadth_filtered_tables/single-cat-filtered-table.qza \ --o-visualization diet_breadth_filtered_tables/single-cat-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Visualize alpha-rarefaction for single diet category subset qiime diversity alpha-rarefaction \ --i-table diet_breadth_filtered_tables/single-cat-filtered-table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 200000 \ --m-metadata-file zoo_metadata_combined.txt \ --o-visualization diet_breadth_filtered_tables/single-cat-alpha-rarefaction.qzv # Calculate beta-diversity metrics for single diet category subset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table diet_breadth_filtered_tables/single-cat-filtered-table.qza \ --p-sampling-depth 89808 \ --m-metadata-file zoo_metadata_combined.txt \ --output-dir diet_breadth_filtered_tables/single-cat-core-metrics-results # Calculate beta-group-significance for conservation status for single diet category subset qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/single-cat-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/single-cat-core-metrics-results/single-cat-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/single-cat-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/single-cat-core-metrics-results/single-cat-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/single-cat-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/single-cat-core-metrics-results/single-cat-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/single-cat-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/single-cat-core-metrics-results/single-cat-weighted-unifrac-conservation-significance.qzv \ --p-pairwise ## Filter for multiple diet category samples qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[NumberDietCategories] IN ('2', '3', '4', '5')" \ --o-filtered-table diet_breadth_filtered_tables/multi-cat-filtered-table.qza # Visualize ASV output statistics for multiple diet category subset qiime feature-table summarize \ --i-table diet_breadth_filtered_tables/multi-cat-filtered-table.qza \ --o-visualization diet_breadth_filtered_tables/multi-cat-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Visualize alpha-rarefaction for multiple diet category subset qiime diversity alpha-rarefaction \ --i-table diet_breadth_filtered_tables/multi-cat-filtered-table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 160000 \ --m-metadata-file zoo_metadata_combined.txt \ --o-visualization diet_breadth_filtered_tables/multi-cat-alpha-rarefaction.qzv # Calculate beta-diversity metrics for multiple diet category subset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table diet_breadth_filtered_tables/multi-cat-filtered-table.qza \ --p-sampling-depth 89808 \ --m-metadata-file zoo_metadata_combined.txt \ --output-dir diet_breadth_filtered_tables/multi-cat-core-metrics-results # Calculate beta-group-significance for conservation status for multiple diet category subset qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/multi-cat-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/multi-cat-core-metrics-results/multi-cat-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/multi-cat-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/multi-cat-core-metrics-results/multi-cat-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/multi-cat-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/multi-cat-core-metrics-results/multi-cat-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix diet_breadth_filtered_tables/multi-cat-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization diet_breadth_filtered_tables/multi-cat-core-metrics-results/multi-cat-weighted-unifrac-conservation-significance.qzv \ --p-pairwise ### Captivity status subset analysis of conservation status ### Subset by “captive_wild” into captive and wild mkdir captivity_filtered_tables ## Filter for captive samples qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[captive_wild]='captive'" \ --o-filtered-table captivity_filtered_tables/capt-filtered-table.qza # Visualize ASV output statistics for captive subset qiime feature-table summarize \ --i-table captivity_filtered_tables/capt-filtered-table.qza \ --o-visualization captivity_filtered_tables/capt-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Visualize alpha-rarefaction for captive subset qiime diversity alpha-rarefaction \ --i-table captivity_filtered_tables/capt-filtered-table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 200000 \ --m-metadata-file zoo_metadata_combined.txt \ --o-visualization captivity_filtered_tables/capt-alpha-rarefaction.qzv # Calculate beta-diversity metrics for captive subset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table captivity_filtered_tables/capt-filtered-table.qza \ --p-sampling-depth 80508 \ --m-metadata-file zoo_metadata_combined.txt \ --output-dir captivity_filtered_tables/capt-core-metrics-results # Calculate beta-group-significance for conservation status for captive subset qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/capt-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/capt-core-metrics-results/capt-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/capt-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/capt-core-metrics-results/capt-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/capt-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/capt-core-metrics-results/capt-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/capt-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/capt-core-metrics-results/capt-weighted-unifrac-conservation-significance.qzv \ --p-pairwise ## Filter for wild samples qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file zoo_metadata_combined.txt \ --p-where "[captive_wild]='wild'" \ --o-filtered-table captivity_filtered_tables/wild-filtered-table.qza # Visualize ASV output statistics for wild subset qiime feature-table summarize \ --i-table captivity_filtered_tables/wild-filtered-table.qza \ --o-visualization captivity_filtered_tables/wild-filtered-table.qzv \ --m-sample-metadata-file zoo_metadata_combined.txt # Visualize alpha-rarefaction for wild subset qiime diversity alpha-rarefaction \ --i-table captivity_filtered_tables/wild-filtered-table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 160000 \ --m-metadata-file zoo_metadata_combined.txt \ --o-visualization captivity_filtered_tables/wild-alpha-rarefaction.qzv # Calculate beta-diversity metrics for wild subset echo Calculating diversity metrics... qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table captivity_filtered_tables/wild-filtered-table.qza \ --p-sampling-depth 80508 \ --m-metadata-file zoo_metadata_combined.txt \ --output-dir captivity_filtered_tables/wild-core-metrics-results # Calculate beta-group-significance for conservation status for wild subset qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/wild-core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/wild-core-metrics-results/wild-jaccard-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/wild-core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/wild-core-metrics-results/wild-bray-curtis-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/wild-core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/wild-core-metrics-results/wild-unweighted-unifrac-conservation-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix captivity_filtered_tables/wild-core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file zoo_metadata_combined.txt \ --m-metadata-column ConservationStatusHMC \ --o-visualization captivity_filtered_tables/wild-core-metrics-results/wild-weighted-unifrac-conservation-significance.qzv \ --p-pairwise echo End of script.