assignment5.sh

#!/bin/bash

#Make sure you're in the folder with METADATA.txt and reads.qza!

qiime deblur denoise-16S --i-demultiplexed-seqs reads.qza --o-table unfiltered_table --o-representative-sequences representative_sequences --o-stats stats.qza --p-trim-length 225 --p-jobs-to-start 4 --p-indel-max 10 --p-sample-stats --verbose

wget https://data.qiime2.org/2019.1/common/gg-13-8-99-nb-classifier.qza
#If you have a large amount of RAM (32GB or greater), try the larger SILVA database:
#wget https://data.qiime2.org/2019.1/common/silva-132-99-nb-classifier.qza

qiime feature-classifier classify-sklearn --i-classifier gg-13-8-99-nb-classifier.qza --i-reads representative_sequences.qza --o-classification taxonomy

#This visualization shows us the sequences/sample spread
qiime feature-table summarize --i-table unfiltered_table.qza --o-visualization table_summary

qiime feature-table filter-samples --i-table unfiltered_table.qza --o-filtered-table table.qza --p-min-frequency 4000
#Taxa bar plots
qiime taxa barplot --i-table unfiltered_table.qza --i-taxonomy taxonomy.qza --m-metadata-file METADATA.txt --o-visualization taxa-bar-plots

#Steps for generating a phylogenetic tree
qiime alignment mafft --i-sequences representative_sequences.qza --o-alignment aligned_representative_sequences

qiime alignment mask --i-alignment aligned_representative_sequences.qza --o-masked-alignment masked_aligned_representative_sequences

qiime phylogeny fasttree --i-alignment masked_aligned_representative_sequences.qza --o-tree unrooted_tree

qiime phylogeny midpoint-root --i-tree unrooted_tree.qza --o-rooted-tree rooted_tree

#Also generates PCoA plots automatically
qiime diversity core-metrics-phylogenetic --i-phylogeny rooted_tree.qza --i-table table.qza --p-sampling-depth 4000 --output-dir diversity_4000 --m-metadata-file METADATA.txt

#Test for between-group differences
qiime diversity alpha-group-significance --i-alpha-diversity diversity_4000/faith_pd_vector.qza --m-metadata-file METADATA.txt --o-visualization diversity_4000/alpha_PD_significance

qiime diversity alpha-group-significance --i-alpha-diversity diversity_4000/shannon_vector.qza --m-metadata-file METADATA.txt --o-visualization diversity_4000/alpha_shannon_significance

#Alpha rarefaction curves show taxon accumulation as a function of sequence depth
qiime diversity alpha-rarefaction --i-table table.qza --p-max-depth 10000 --o-visualization diversity_4000/alpha_rarefaction.qzv --m-metadata-file METADATA.txt --i-phylogeny rooted_tree.qza