#feburary 27, 2023 # !/bin/bash #locate parkinsons datasets #pathway is /mnt/datasets/project_2/parkinsons # Create a directory for your project and navigate to it mkdir /data/team3_parkinsons cd /data/team3_parkinsons # Import data while working directory is `/data/team3_parkinsons` #use manifest version bc theres a manifest file there?? qiime tools import \ --type "SampleData[SequencesWithQuality]" \ --input-format SingleEndFastqManifestPhred33V2 \ --input-path /mnt/datasets/project_2/parkinsons/parkinsons_manifest.txt \ --output-path ./demux_seqs.qza #This code both imports and demultiplexes your dataset. (this means you won't need to run a separate demultiplexing step) #send to your local computer #visualize demux_seqs.qza to .qzv to figure out rarefaction cutoffs # Create visualization of demultiplexed samples qiime demux summarize \ --i-data demux_seqs.qza \ --o-visualization demux_seqs.qzv #send to your local computer ###Denoising and Clustering # Determine ASVs with DADA2 qiime dada2 denoise-single \ --i-demultiplexed-seqs demux_seqs.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 (might not be needed → like qza yes, but qzv stuff) qiime metadata tabulate \ --m-input-file stats.qza \ --o-visualization stats.qzv # Visualize ASVs stats (might not be needed → like qza yes, but qzv stuff) qiime feature-table summarize \ --i-table table.qza \ --o-visualization table.qzv \ --m-sample-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt #feature table → (might not be needed → like qza yes, but qzv stuff) qiime feature-table tabulate-seqs \ --i-data rep-seqs.qza \ --o-visualization rep-seqs.qzv #March 20, 2023 #visualize PD filtered table qiime feature-table summarize \ --i-table pd-filtered-table.qza \ --o-visualization pd-filtered-table.qzv \ --m-sample-metadata-file /data/pdt3/metadata1.txt \ #filter out mitochondria qiime taxa filter-table \ --i-table pd-filtered-table.qza \ --i-taxonomy taxonomy.qza \ --p-exclude mitochondria,chloroplast \ --o-filtered-table pd-nomitochondria.qza #view qiime feature-table summarize \ --i-table pd-nomitochondria.qza \ --o-visualization table-no-mitochondria-no-chloroplast.qzv \ --m-sample-metadata-file /data/pdt3/metadata1.txt \ # Visualize taxonomy qiime metadata tabulate \ --m-input-file taxonomy.qza \ --o-visualization taxonomy.qzv #March 22, 2023 # Taxonomy barplots qiime taxa barplot \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-visualization taxa-bar-plots-not.qzv ## Metadata-based filtering qiime feature-table filter-samples \ --i-table table-no-mitochondria-no-chloroplast.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-filtered-table pd-filtered-table.qza #visualize PD filtered table qiime feature-table summarize \ --i-table pd-filtered-table.qza \ --o-visualization pd-filtered-table.qzv \ --m-sample-metadata-file /data/pdt3/metadata1.txt \ #filter out mitochondria qiime taxa filter-table \ --i-table pd-filtered-table.qza \ --i-taxonomy taxonomy.qza \ --p-exclude mitochondria,chloroplast \ --o-filtered-table pd-nomitochondria.qza #view qiime feature-table summarize \ --i-table pd-nomitochondria.qza \ --o-visualization table-no-mitochondria-no-chloroplast.qzv \ --m-sample-metadata-file /data/pdt3/metadata1.txt \ # Visualize taxonomy qiime metadata tabulate \ --m-input-file taxonomy.qza \ --o-visualization taxonomy.qzv # Generate a tree for phylogenetic diversity analyses 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 # Alpha-rarefaction qiime diversity alpha-rarefaction \ --i-table table-no-mitochondria-no-chloroplast.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 7563 \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-visualization alpha-rarefaction.qzv # Calculate alpha- and beta-diversity metrics qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table-no-mitochondria-no-chloroplast.qza \ --p-sampling-depth 7563\ --m-metadata-file /data/pdt3/metadata1.txt \ --output-dir meta3 Waittt for the alphas # Calculate alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity meta3/faith_pd_vector.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-visualization visual/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity meta3/evenness_vector.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-visualization visual/evenness-group-significance.qzv Beta diversity for depressed qiime diversity beta-group-significance \ --i-distance-matrix meta3/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --m-metadata-column Combined \ --o-visualization meta3/unweighted-unifrac-depressed-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix meta3/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --m-metadata-column Combined \ --o-visualization meta3/unweighted-unifrac-depressed-group-significance.qzv \ --p-pairwise # Principal Coordinates Analysis (PCoA) - Unweighted Unifrac & Bray Curtis qiime emperor plot \ --i-pcoa meta3/unweighted_unifrac_pcoa_results.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-visualization meta3/depressed-unweighted-unifrac-emperor.qzv qiime emperor plot \ --i-pcoa meta3/bray_curtis_pcoa_results.qza \ --m-metadata-file /data/pdt3/metadata1.txt \ --o-visualization meta3/pd-bray-curtis-emperor.qzv #To export files (working in new directory export) qiime tools export \ --input-path /data/pdt3/project2/meta3/pd-bray-curtis-emperor.qzv \ --output-path pd-bray-curtis-emperor_export qiime tools export \ --input-path /data/pdt3/project2/taxonomy.qza \ --output-path taxonomy_export qiime tools export \ --input-path /data/pdt3/project2/rep-seqs.qza \ --output-path rep-seqs_export qiime tools export \ --input-path /data/pdt3/project2/table.qza \ --output-path table_export \ qiime tools export \ --input-path /data/pdt3/project2/rooted-tree.qza\ --output-path rooted-tree_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/weighted_unifrac_distance_matrix.qza\ --output-path weighted_unifrac_distance_matrix_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/unweighted_unifrac_distance_matrix.qza\ --output-path unweighted_unifrac_distance_matrix_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/rarefied_table.qza\ --output-path rarefied_table_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/observed_features_vector.qza\ --output-path observed_features_vector_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/shannon_vector.qza\ --output-path shannon_vector_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/weighted_unifrac_distance_matrix.qza\ --output-path weighted_unifrac_distance_matrix_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/weighted_unifrac_emperor.qzv\ --output-path weighted_unifrac_emperor_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/weighted_unifrac_pcoa_results.qza\ --output-path weighted_unifrac_pcoa_results_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/bray_curtis_distance_matrix.qza\ --output-path bray_curtis_distance_matrix_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/bray_curtis_emperor.qzv\ --output-path bray_curtis_emperor_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/bray_curtis_pcoa_results.qza\ --output-path bray_curtis_pcoa_results_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/evenness-group-significance.qza\ --output-path evenness-group-significance_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/evenness_vector.qza\ --output-path evenness_vector_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/faith-pd-group-significance.qzv\ --output-path faith-pd-group-significance_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/faith_pd_vector.qza\ --output-path faith_pd_vector_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/jaccard_distance_matrix.qza\ --output-path jaccard_distance_matrix_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/jaccard_emperor.qzv\ --output-path jaccard_emperor_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/jaccard_pcoa_results.qza\ --output-path jaccard_pcoa_results_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/unweighted-unifrac-depressed-significance.qzv\ --output-path unweighted-unifrac-depressed-significance_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/unweighted_unifrac_distance_matrix.qza\ --output-path unweighted_unifrac_distance_matrix_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/unweighted_unifrac_emperor.qzv\ --output-path unweighted_unifrac_emperor_export \ qiime tools export \ --input-path /data/pdt3/project2/meta3/unweighted_unifrac_pcoa_results.qza\ --output-path unweighted_unifrac_pcoa_results_export \