## QIIME2 Aim 1 Script #making a directory dedicated to project 2 cd /data mkdir parkinsons cd parkinsons #importing and demultiplexing --type "SampleData[SequencesWithQuality]" \ --input-format SingleEndFastqManifestPhred33V2 \ --input-path /mnt/datasets/project_2/parkinsons/parkinsons_manifest.txt \ --output-path /data/parkinsons/demux_seqs.qza #Visualing the demux samples qiime demux summarize \ --i-data demux_seqs.qza \ --o-visualization demux.qzv #moving demux.qzv to my personal computer scp root@10.19.139.171:/data/parkinsons/demux.qzv . #Denoising our sequences and truncation to 251 (determining ASVs with DADA2 ) qiime dada2 denoise-single \ --i-demultiplexed-seqs demux_seqs.qza \ --p-trunc-len 251 \ --o-table dada2_table.qza \ --o-representative-sequences dada2_rep_set.qza \ --o-denoising-stats dada2_stats.qza. #Create a visualization for the data after denoising qiime metadata tabulate \ --m-input-file dada2_stats.qza \ --o-visualization dada2_stats.qzv #Creating the Feature Table qiime feature-table summarize \ --i-table dada2_table.qza \ --o-visualization dada2_table.qzv \ --m-sample-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt qiime feature-table tabulate-seqs \ --i-data dada2_rep_set.qza \ --o-visualization dada2_rep_set.qzv #moving dada2_table.qzv to my personal computer scp root@10.19.139.171:/data/parkinsons/dada2_table.qzv . #moving dada2_rep_set.qzv to my personal computer scp root@10.19.139.171:/data/parkinsons/dada2_rep_set.qzv . #Taxonomic analysis qiime feature-classifier classify-sklearn \ --i-classifier /mnt/datasets/classifiers/silva-138-99-515-806-nb-classifier.qza \ --i-reads dada2_rep_set.qza \ --o-classification taxonomy.qza qiime metadata tabulate \ --m-input-file taxonomy.qza \ --o-visualization taxonomy.qzv #Taxonomy barplots (non-filtered) qiime taxa barplot \ --i-table dada2_table.qza \ --i-taxonomy taxonomy.qza \ --m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \ --o-visualization taxa-bar-plots.qzv #moving taxonomy.qzv to my personal computer scp root@10.19.139.171:/data/parkinsons/taxonomy.qzv . #moving taxa-bar-plots.qzv to my personal computer scp root@10.19.139.171:/data/parkinsons/taxa-bar-plots.qzv . qiime feature-table summarize \ --i-table table-no-mitochondria-no-chloroplast.qza \ --o-visualization table-no-mitochondria-no-chloroplast.qzv \ --m-sample-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt #moving table-no-mitochondria-no-chloroplast.qzv to my personal computer scp root@10.19.139.171:/data/parkinsons/table-no-mitochondria-no-chloroplast.qzv . #Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences dada2_rep_set.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 #Calculating 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 4000 \ --m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \ --output-dir core-metrics-results_new #Calculating alpha-group-significance qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results_new/faith_pd_vector.qza \ --m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \ --o-visualization core-metrics-results_new/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results_new/evenness_vector.qza \ --m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \ --o-visualization core-metrics-results_new/evenness-group-significance.qzv #Moving alpha diversity generated files to my personal computer scp root@10.19.139.171:/data/parkinsons/core-metrics-results_new/evenness-group-significance.qzv . scp root@10.19.139.171:/data/parkinsons/core-metrics-results_new/faith-pd-group-significance.qzv . #Moving beta diversity generated files to my personal computer scp root@10.19.139.171:/data/parkinsons/core-metrics-results_new/weighted_unifrac_emperor.qzv . scp root@10.19.139.171:/data/parkinsons/core-metrics-results_new/jaccard_emperor.qzv . scp root@10.19.139.171:/data/parkinsons/core-metrics-results_new/bray_curtis_emperor.qzv . scp root@10.19.139.171:/data/parkinsons/core-metrics-results_new/unweighted_unifrac_emperor.qzv . #Exporting the qza files qiime tools export \ --input-path rooted-tree.qza \ --output-path rooted_tree_export qiime tools export \ --input-path core-metrics-results_new/weighted_unifrac_distance_matrix.qza \ --output-path weighted_unifrac_distance_matrix_export qiime tools export \ --input-path dada2_rep_set.qza \ --output-path rep-set_export qiime tools export \ --input-path taxonomy.qza \ --output-path taxonomy_export qiime tools export \ --input-path rooted-tree.qza \ --output-path rooted-tree_export qiime tools export \ --input-path unrooted-tree.qza \ --output-path unrooted-tree_export qiime tools export \ --input-path unrooted-tree.qza \ --output-path unrooted-tree_export qiime tools export \ --input-path aligned-rep-seqs.qza \ --output-path aligned-rep-seqs_export qiime tools export \ --input-path dada2_stats.qza \ --output-path dada2_stats_export qiime tools export \ --input-path demux_seqs.qza \ --output-path demux_seqs_export qiime tools export \ --input-path table-no-mitochondria-no-chloroplast.qza \ --output-path table-no-mitochondria-no-chloroplast_export #Moving the generated export files to my personal computer scp root@10.19.139.171:/data/parkinsons/rep-set_export/dna-sequences.fasta . scp root@10.19.139.171:/data/parkinsons/weighted_unifrac_distance_matrix_export/distance-matrix.tsv . scp root@10.19.139.171:/data/parkinsons/rooted-tree_export/tree.nwk . scp root@10.19.139.171:/data/parkinsons/unrooted-tree_export/tree.nwk . scp root@10.19.139.171:/data/parkinsons/taxonomy_export/taxonomy.tsv . scp root@10.19.139.171:/data/parkinsons/dada2_stats_export/stats.tsv . #Exporting a biom file into a text file biom convert -i table-no-mitochondria-no-chloroplast_export/feature-table.biom --to-tsv -o feature-table_new.txt #Moving feature-table_new.txt to my personal computer scp root@10.19.139.171:/data/parkinsons/feature-table_new.txt .