#!/bin/bash ### QIME2 MICROBIOME SEQUENCE LIBARY IMPORTING AND MANIPULATION # Create a directory for your project mkdir /data/fish_data # Navigate to fish folder cd /mnt/datasets/project_2/fish # Import and demultiplex midgut_manifest file in /mnt/datasets/project_2/fish qiime tools import \ --type "SampleData[SequencesWithQuality]" \ --input-format SingleEndFastqManifestPhred33V2 \ --input-path /data/fish_data/midgut_manifest.txt \ --output-path /data/fish_data/midgut_demux.qza # Import and demultiplex hindgut_manifest file in /mnt/datasets/project_2/fish qiime tools import \ --type "SampleData[SequencesWithQuality]" \ --input-format SingleEndFastqManifestPhred33V2 \ --input-path /data/fish_data/hindgut_manifest.txt \ --output-path /data/fish_data/hindgut_demux.qza # Navigate to fish_data folder cd /data/fish_data # Create visualization of demultiplexed midgut demux.qza files qiime demux summarize \ --i-data midgut_demux.qza \ --o-visualization midgut_demux.qzv # Create visualization of demultiplexed hindgut demux.qza files qiime demux summarize \ --i-data hindgut_demux.qza \ --o-visualization hindgut_demux.qzv # Make directory in home directory on local machine mkdir project_2 cd project_2 # Denoise using the selected truncation length # Working directory is 'fish_data' qiime dada2 denoise-single \ --i-demultiplexed-seqs midgut_demux.qza \ --p-trim-left 0 \ --p-trunc-len 180 \ --o-representative-sequences midgut_rep_seqs.qza \ --o-table midgut_table.qza \ --o-denoising-stats midgut_stats.qza qiime dada2 denoise-single \ --i-demultiplexed-seqs hindgut_demux.qza \ --p-trim-left 0 \ --p-trunc-len 180 \ --o-representative-sequences hindgut_rep_seqs.qza \ --o-table hindgut_table.qza \ --o-denoising-stats hindgut_stats.qza # Visualize DADA2 stats qiime metadata tabulate \ --m-input-file midgut_stats.qza \ --o-visualization midgut_stats.qzv qiime metadata tabulate \ --m-input-file hindgut_stats.qza \ --o-visualization hindgut_stats.qzv # Visualize ASVs stats # working directory is `fish_data` qiime feature-table summarize \ --i-table /data/fish_data/midgut_table.qza \ --o-visualization /data/fish_data/midgut_table.qzv \ qiime feature-table summarize \ --i-table midgut_table.qza \ --o-visualization midgut_table.qzv \ qiime feature-table tabulate-seqs \ --i-data midgut_rep_seqs.qza \ --o-visualization midgut_rep_seqs.qzv qiime feature-table summarize \ --i-table hindgut_table.qza \ --o-visualization hindgut_table.qzv \ qiime feature-table tabulate-seqs \ --i-data hindgut_rep_seqs.qza \ --o-visualization hindgut_rep_seqs.qzv # Retrain the classifier qiime feature-classifier extract-reads \ --i-sequences /mnt/datasets/silva_ref_files/silva-138-99-seqs.qza \ --p-f-primer GTGYCAGCMGCCGCGGTAA \ # f and r primers obtained from github --p-r-primer GGACTACNVGGGTWTCTAAT \ --p-trunc-len 180 \ --o-reads ref_seqs_trimmed.qza # Train classifier with your new ref-seq file qiime feature-classifier fit-classifier-naive-bayes \ --i-reference-reads ref_seqs_trimmed.qza \ --i-reference-taxonomy /mnt/datasets/silva_ref_files/silva-138-99-tax.qza \ --o-classifier classifier.qza # Use the trained classifier to assign taxonomy to your reads (rep-seqs.qza) qiime feature-classifier classify-sklearn \ --i-classifier /mnt/datasets/classifiers/silva-138-99-515-806-nb-classifier.qza \ --i-reads midgut_rep_seqs.qza \ --o-classification midgut_taxonomy.qza qiime feature-classifier classify-sklearn \ --i-classifier /mnt/datasets/classifiers/silva-138-99-515-806-nb-classifier.qza \ --i-reads hindgut_rep_seqs.qza \ --o-classification hindgut_taxonomy.qza # Create visualization taxonomy files qiime metadata tabulate \ --m-input-file midgut_taxonomy.qza \ --o-visualization midgut_taxonomy.qzv qiime metadata tabulate \ --m-input-file hindgut_taxonomy.qza \ --o-visualization hindgut_taxonomy.qzv #Taxonomy barplots qiime taxa barplot \ --i-table /data/fish_data/midgut_table.qza \ --i-taxonomy /data/fish_data/midgut_taxonomy.qza \ --o-visualization /data/fish_data/midgut-taxa-bar-plots.qzv qiime taxa barplot \ --i-table /data/fish_data/hindgut_table.qza \ --i-taxonomy /data/fish_data/hindgut_taxonomy.qza \ --o-visualization /data/fish_data/hindgut-taxa-bar-plots.qzv # Generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences midgut_rep_seqs.qza \ --o-alignment midgut-aligned-rep-seqs.qza \ --o-masked-alignment midgut-masked-aligned-rep-seqs.qza \ --o-tree midgut-unrooted-tree.qza \ --o-rooted-tree midgut-rooted-tree.qza qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences hindgut_rep_seqs.qza \ --o-alignment hindgut-aligned-rep-seqs.qza \ --o-masked-alignment hindgut-masked-aligned-rep-seqs.qza \ --o-tree hindgut-unrooted-tree.qza \ --o-rooted-tree hindgut-rooted-tree.qza ### EXPORT FILES FOR DIVERSITY ANALYSES AND DIFFERENTIAL ABUNDANCE # MIDGUT qiime tools export \ --input-path midgut_table.qza \ --output-path midgut_table_export qiime tools export \ --input-path midgut_taxonomy.qza \ --output-path midgut_taxonomy_export qiime tools export \ --input-path midgut-rooted-tree.qza \ --output-path midgut_rooted_tree_export # HINDGUT qiime tools export \ --input-path hindgut_table.qza \ --output-path hindgut_table_export qiime tools export \ --input-path hindgut_taxonomy.qza \ --output-path hindgut_taxonomy_export qiime tools export \ --input-path hindgut-rooted-tree.qza \ --output-path hindgut_rooted_tree_export #To export a biom file into a text file biom convert -i feature-table.biom --to-tsv -o midgut_table.txt biom convert -i feature-table.biom --to-tsv -o hindgut_table.txt