#Project 2 checklist QIIME #Claudia March 5th (DONE) #logging into team 10 server ssh root@10.19.139.155 #making a directory dedicated to project 2 cd /data mkdir project_2 cd project_2 # importing and demultiplexing qiime tools import \ --type 'SampleData[SequencesWithQuality]' \ --input-path /mnt/datasets/project_2/parkinsons/parkinsons_manifest.txt \ --input-format SingleEndFastqManifestPhred33V2 \ --output-path demux.qza #Visualing the demux samples qiime demux summarize \ --i-data demux.qza \ --o-visualization demux.qzv #moving demux.qzv to my personal computer scp root@10.19.139.155:/data/project_2/demux.qzv . #Parsa March 13th #moving new metadata file from my personal server to our team server scp metadata.tsv root@10.19.139.155:/data/project_2 #Steven March 13th #determining ASVs with DADA2 qiime dada2 denoise-single \ --i-demultiplexed-seqs demux.qza \ --p-trim-left 0 \ --p-trunc-len 251 \ --o-representative-sequences rep-seqs.qza \ --o-table table.qza \ --o-denoising-stats stats.qza # Visualize DADA2 stats qiime metadata tabulate \ --m-input-file stats.qza \ --o-visualization stats.qzv # Visualize ASVs stats qiime feature-table summarize \ --i-table table.qza \ --o-visualization table.qzv \ --m-sample-metadata-file /root/data/project_2/metadata.tsv qiime feature-table tabulate-seqs \ --i-data rep-seqs.qza \ --o-visualization rep-seqs.qzv #Claudia March 15th 2:34 pm # Taxonomic analysis 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 qiime taxa barplot \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --m-metadata-file metadata.tsv \ --o-visualization taxa-bar-plots.qzv 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 #Steven March 16 qiime feature-table filter-samples --i-table table-no-mitochondria-no-chloroplast.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[Disease]='PD'" --o-filtered-table PD-only-table.qza #making a directory dedicated to fatigue metadata cd /data/project_2 mkdir fatigue_metadata cd fatigue_metadata #making a directory dedicated to sleep problems metadata cd /data/project_2 mkdir sleep_problems_metadata cd sleep_problems_metadata #making a directory dedicated to antidepressant use metadata cd /data/project_2 mkdir antidepressant_use_metadata cd antidepressant_use_metadata #making a directory dedicated to apathy metadata cd /data/project_2 mkdir apathy_binary_metadata cd apathy_binary_metadata # ---------------------------------------------------------------FATIGUE ONLY CODE---------------------------------------------------------------------- #Parsa March 16th 11:57 PM qiime feature-table filter-samples --i-table /root/data/project_2/PD-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[fatigue_binary] IN ('yes', 'no')" --o-filtered-table NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza --i-table NA-filtered-out-table.qza --p-sampling-depth 6000 --m-metadata-file /root/data/project_2/metadata.tsv --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/faith_pd_vector.qza --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/evenness_vector.qza --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/unweighted-unifrac-fatigue-significance.qzv --p-pairwise #Making more box plots to see beta diversity significance qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/weighted-unifrac-fatigue-significance.qzv --p-pairwise qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/bray_curtis_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/bray-curtis-fatigue-significance.qzv --p-pairwise qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/jaccard_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/jaccard-fatigue-significance.qzv --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction --i-table /root/data/project_2/fatigue_metadata/NA-filtered-out-table.qza --i-phylogeny /root/data/project_2/fatigue_metadata/rooted-tree.qza --p-max-depth 24026 --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization alpha-rarefaction-2.qzv # ---------------------------------------------------------------Apathy ONLY CODE---------------------------------------------------------------------- #Steven March 18, 2022 #Filtering for apathy qiime feature-table filter-samples --i-table /root/data/project_2/PD-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[apathy_binary] IN ('yes', 'no')" --o-filtered-table apathy-NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table apathy-NA-filtered-out-table.qza \ --p-sampling-depth 8000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/apathy_binary_metadata/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/apathy_binary_metadata/core-metrics-results/unweighted-unifrac-apathy-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/apathy_binary_metadata/core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/apathy_binary_metadata/core-metrics-results/weighted-unifrac-apathy-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/apathy_binary_metadata/core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/apathy_binary_metadata/core-metrics-results/bray-curtis-apathy-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/apathy_binary_metadata/core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/apathy_binary_metadata/core-metrics-results/jaccard-apathy-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/apathy_binary_metadata/apathy-NA-filtered-out-table.qza \ --i-phylogeny /root/data/project_2/apathy_binary_metadata/rooted-tree.qza \ --p-max-depth 21491 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization alpha-rarefaction.qzv # ---------------------------------------------------------------Sleep problems ONLY CODE---------------------------------------------------------------------- #Baria March 18, 2022 #Filtering for sleep problems qiime feature-table filter-samples --i-table /root/data/project_2/PD-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[sleep_problems] IN ('yes', 'no')" --o-filtered-table sleep_problems-NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table sleep_problems-NA-filtered-out-table.qza \ --p-sampling-depth 6000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/sleep_problems_metadata/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/sleep_problems_metadata/core-metrics-results/unweighted-unifrac-sleep_problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/sleep_problems_metadata/core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/sleep_problems_metadata/core-metrics-results/weighted-unifrac-sleep_problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/sleep_problems_metadata/core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/sleep_problems_metadata/core-metrics-results/bray-curtis-sleep-problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/sleep_problems_metadata/core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/sleep_problems_metadata/core-metrics-results/jaccard-sleep-problems-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/control_samples/sleep_problems_metadata/sleep_problems-NA-filtered-out-table.qza \ --i-phylogeny /root/data/project_2/control_samples/sleep_problems_metadata/rooted-tree.qza \ --p-max-depth 24026 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization alpha-rarefaction.qzv # ---------------------------------------------------------------Antidepressant use ONLY CODE---------------------------------------------------------------------- #filtering out NA values in the antidepressant column qiime feature-table filter-samples \ --i-table /root/data/project_2/PD-only-table.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --p-where "[antidepressant_use] IN ('yes', 'no')" \ --o-filtered-table antidep-NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table antidep-NA-filtered-out-table.qza \ --p-sampling-depth 8000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/antidepressant_use_metadata/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column antidepressant_use \ --o-visualization /root/data/project_2/antidepressant_use_metadata/core-metrics-results/unweighted-unifrac-antidep-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/antidepressant_use_metadata/antidep-NA-filtered-out-table.qza \ --i-phylogeny /root/data/project_2/antidepressant_use_metadata/rooted-tree.qza \ --p-max-depth 12109 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization /root/data/project_2/antidepressant_use_metadata/alpha-rarefaction.qzv # ------------------------------------------------changing sampling depth to 6000 for Antidepressant use---------------------------------------------------------------------- #making new directory for the new sampling depth attempt mkdir antidep_6000 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table /root/data/project_2/antidepressant_use_metadata/antidep-NA-filtered-out-table.qza \ --p-sampling-depth 6000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/antidep_6000/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column antidepressant_use \ --o-visualization /root/data/project_2/antidep_6000/core-metrics-results/unweighted-unifrac-antidep-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/antidep_6000/core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column antidepressant_use \ --o-visualization /root/data/project_2/antidep_6000/core-metrics-results/weighted-unifrac-antidep-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/antidep_6000/core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column antidepressant_use \ --o-visualization /root/data/project_2/antidep_6000/core-metrics-results/bray-curtis-antidep-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/antidep_6000/core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column antidepressant_use \ --o-visualization /root/data/project_2/antidep_6000/core-metrics-results/jaccard-antidep-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/antidepressant_use_metadata/antidep-NA-filtered-out-table.qza \ --i-phylogeny /root/data/project_2/antidepressant_use_metadata/rooted-tree.qza \ --p-max-depth 24026 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization /root/data/project_2/antidep_6000/alpha-rarefaction.qzv ------------------------------------------------------------------------------------------------------------------------------------------------- #Parsa 28 Mar 2022 (4:58 PM) #exporting data for differential abundance analysis #exporting table.qza , taxonomy.qza and rooted-tree.qza qiime tools export \ --input-path table.qza \ --output-path exported qiime tools export \ --input-path taxonomy.qza \ --output-path exported qiime tools export \ --input-path rooted-tree.qza \ --output-path exported # Edit the column names exported/taxonomy.tsv sed \ '1 s/Feature ID/#OTUID/;1 s/Taxon/taxonomy/;1 s/Confidence/confidence/' \ exported/taxonomy.tsv \ > exported/biom-taxonomy.tsv #combine taxonomy with biome data biom add-metadata \ -i exported/feature-table.biom \ -o exported/table-with-taxonomy.biom \ --observation-metadata-fp exported/biom-taxonomy.tsv \ --sc-separated taxonomy -----------------------------------------------------------CONTROL SAMPLES ANALYSIS------------------------------------------------------------------------------------- # Claudia March 28th 7:13 pm #Control samples analysis #Copying the no mitochondria and no chloroplast table cp table-no-mitochondria-no-chloroplast.qza /root/data/project_2/control_samples/table_no_mitochondria_no_chloroplast.qza #Filtering only control samples qiime feature-table filter-samples --i-table table-no-mitochondria-no-chloroplast.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[Disease]=‘Control’” --o-filtered-table Control-only-table.qza #making a directory dedicated to each metadata category mkdir fatigue_metadata cd /data/project_2/control_samples mkdir sleep_problems_metadata cd /data/project_2/control_samples mkdir antidepressant_use_metadata cd /data/project_2/control_samples mkdir apathy_binary_metadata cd /data/project_2/control_samples # Baria March 29th 12:07 PM #SLEEP PROBLEMS in CONTROL PATIENTS #Filtering for sleep problems qiime feature-table filter-samples --i-table /root/data/project_2/control_samples/Control-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[sleep_problems] IN ('yes', 'no')" --o-filtered-table sleep_problems-NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table sleep_problems-NA-filtered-out-table.qza \ --p-sampling-depth 6000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/unweighted-unifrac-sleep_problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/weighted-unifrac-sleep_problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/bray-curtis-sleep-problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/control_samples/sleep_problems_metadata/core-metrics-results/jaccard-sleep-problems-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/sleep_problems_metadata/sleep_problems-NA-filtered-out-table.qza \ --i-phylogeny /root/data/project_2/sleep_problems_metadata/rooted-tree.qza \ --p-max-depth 24026 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization alpha-rarefaction.qzv #APATHY in CONTROL PATIENTS #Filtering for apathy qiime feature-table filter-samples --i-table /root/data/project_2/PD-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[apathy_binary] IN ('yes', 'no')" --o-filtered-table apathy-NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table apathy-NA-filtered-out-table.qza \ --p-sampling-depth 6000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/unweighted-unifrac-apathy-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/weighted-unifrac-apathy-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/bray-curtis-apathy-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/control_samples/apathy_binary_metadata/core-metrics-results/jaccard-apathy-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/control_samples/apathy_binary_metadata/apathy-NA-filtered-out-table.qza \ --i-phylogeny /root/data//project_2/control_samples/apathy_binary_metadata/rooted-tree.qza \ --p-max-depth 24026 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization alpha-rarefaction.qzv #ANTIDEPRESSANT USE in CONTROL PATIENTS #Filtering for antidepressant use qiime feature-table filter-samples --i-table /root/data/project_2/PD-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[antidepressant_use] IN ('yes', 'no')" --o-filtered-table antidep-NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table antidep-NA-filtered-out-table.qza \ --p-sampling-depth 6000 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/unweighted-unifrac-sleep_problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column sleep_problems \ --o-visualization /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/weighted-unifrac-sleep_problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/bray_curtis_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/bray-curtis-sleep-problems-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/jaccard_distance_matrix.qza \ --m-metadata-file /root/data/project_2/metadata.tsv \ --m-metadata-column apathy_binary \ --o-visualization /root/data/project_2/control_samples/antidepressant_use_metadata/core-metrics-results/jaccard-sleep-problems-significance.qzv \ --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table /root/data/project_2/control_samples/antidepressant_use_metadata/apathy-NA-filtered-out-table.qza \ --i-phylogeny /root/data/project_2/control_samples/antidepressant_use_metadata/rooted-tree.qza \ --p-max-depth 24026 \ --m-metadata-file /root/data/project_2/metadata.tsv \ --o-visualization alpha-rarefaction.qzv # ---------------------------------------------------------------FATIGUE ONLY CODE---------------------------------------------------------------------- #Claudia March 2 qiime feature-table filter-samples --i-table /root/data/project_2/PD-only-table.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[fatigue_binary] IN ('yes', 'no')" --o-filtered-table NA-filtered-out-table.qza #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences /root/data/project_2/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 # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza --i-table NA-filtered-out-table.qza --p-sampling-depth 6000 --m-metadata-file /root/data/project_2/metadata.tsv --output-dir core-metrics-results # Calculate alpha-group-significance qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/faith_pd_vector.qza --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance --i-alpha-diversity core-metrics-results/evenness_vector.qza --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization core-metrics-results/evenness-group-significance.qzv # Calculate beta-group-significance qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/unweighted-unifrac-fatigue-significance.qzv --p-pairwise #Making more box plots to see beta diversity significance qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/weighted-unifrac-fatigue-significance.qzv --p-pairwise qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/bray_curtis_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/bray-curtis-fatigue-significance.qzv --p-pairwise qiime diversity beta-group-significance --i-distance-matrix core-metrics-results/jaccard_distance_matrix.qza --m-metadata-file /root/data/project_2/metadata.tsv --m-metadata-column fatigue_binary --o-visualization core-metrics-results/jaccard-fatigue-significance.qzv --p-pairwise # Alpha-rarefaction qiime diversity alpha-rarefaction --i-table /root/data/project_2/fatigue_metadata/NA-filtered-out-table.qza --i-phylogeny /root/data/project_2/fatigue_metadata/rooted-tree.qza --p-max-depth 24026 --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization alpha-rarefaction-2.qzv -------------------------------------------------------------------------FIGURE 1 CODING------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ #Creating a taxa-bar-plot with PD patients only information qiime taxa barplot --i-table PD-only-table.qza --i-taxonomy taxonomy.qza --m-metadata-file metadata.tsv --o-visualization taxa-bar-plots-PD-only.qzv #Creating a taxa-bar-plot with Control patients only information qiime taxa barplot --i-table Control-only-table.qza --i-taxonomy /root/data/project_2/taxonomy.qza --m-metadata-file /root/data/project_2/metadata.tsv --o-visualization taxa-bar-plots-control-only.qzv ------------------------------------------------------------------------Indicator taxa coding --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- qiime feature-table filter-samples --i-table table-no-mitochondria-no-chloroplast.qza --m-metadata-file /root/data/project_2/metadata.tsv --p-where "[antidepressant_use] IN ('yes', 'no')" --o-filtered-table antidep-NA-filtered-out-both-PD-and-control-table.qza