# Increased body mass is associated with decreased gut microbiome diversity in Parkinson’s Disease patients

# Novia Chan, Clarisse Echavez, Angela Mathews, Josie Setiawan
# Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

## Supplemental QIIME2 Script

## Open a Command Prompt window and sign in

## Create directory for Parkinson's project
mkdir /data/parkinsons_metadata

## Import and Demultiplex while directory is '/data/parkinsons_metadata'
qiime tools import \
--type 'SampleData[SequencesWithQuality]' \
--input-path /mnt/datasets/project_2/parkinsons/parkinsons_manifest.txt \
--input-format SingleEndFastqManifestPhred33V2 \
--output-path parkinsons_demux.qza

# Create visualization of demultiplexed samples
qiime demux summarize \
--i-data parkinsons_demux.qza \
--o-visualization parkinsons_demux.qzv

## Visualize parkinsons_demux.qzv on local machine
scp root@<IP>:/data/parkinsons_metadata/parkinsons_demux.qzv .

## Transfer sorted Parkinson's metadata file from local directory to team server
# In the directory that contains the parkinsons_metadata_sorted_grouped.tsv on the local machine
scp parkinsons_metadata_sorted_grouped.tsv root@<IP>:/data/parkinsons_metadata

## Determining ASVs
# Determine ASVs with DADA2
qiime dada2 denoise-single \
--i-demultiplexed-seqs parkinsons_demux.qza \
--p-trim-left 0 \
--p-trunc-len 251 \
--o-representative-sequences parkinsons-rep-seqs.qza \
--o-table parkinsons-table.qza \
--o-denoising-stats parkinsons-stats.qza

# Generating FeatureTable and FeatureData summaries
# Visualize ASVs stats
qiime feature-table summarize \
--i-table parkinsons-table.qza \
--o-visualization parkinsons-table.qzv \
--m-sample-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt

qiime feature-table tabulate-seqs \
--i-data parkinsons-rep-seqs.qza \
--o-visualization parkinsons-rep-seqs.qzv

# Visualize DADA2 stats
qiime metadata tabulate \
--m-input-file parkinsons-stats.qza \
--o-visualization parkinsons-stats.qzv

## Taxonomic Analysis
# Assign taxonomy 
qiime feature-classifier classify-sklearn \
--i-classifier /mnt/datasets/classifiers/silva-138-99-515-806-nb-classifier.qza \
--i-reads parkinsons-rep-seqs.qza \
--o-classification parkinsons-taxonomy.qza

# Visualize taxonomy 
qiime metadata tabulate \
--m-input-file parkinsons-taxonomy.qza \
--o-visualization parkinsons-taxonomy.qzv

# Taxonomy barplots (non-filtered)
qiime taxa barplot \
--i-table parkinsons-table.qza \
--i-taxonomy parkinsons-taxonomy.qza \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--o-visualization taxa-bar-plots.qzv

# Taxonomy barplots (Control-filtered)
qiime taxa barplot \
--i-table control-filtered-table.qza \
--i-taxonomy parkinsons-taxonomy.qza \
--m-metadata-file parkinsons_metadata_sorted_grouped.tsv \
--o-visualization control-filtered-taxa-bar-plots.qzv

# Taxonomy barplots (PD-filtered)
qiime taxa barplot \
--i-table pd-filtered-table.qza \
--i-taxonomy parkinsons-taxonomy.qza \
--m-metadata-file parkinsons_metadata_sorted_grouped.tsv \
--o-visualization pd-filtered-taxa-bar-plots.qzv

# Generating tree for phylogenetic diversity analyses
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences parkinsons-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

## Metadata-based filtering
# Filter metadata for keeping only PD
qiime feature-table filter-samples \
--i-table parkinsons-table-2.qza \
--m-metadata-file parkinsons_metadata_sorted_grouped.tsv \
--p-where "[Disease]='PD'" \
--o-filtered-table pd-filtered-table.qza

# Filter metadata for keeping on Control
qiime feature-table filter-samples \
--i-table parkinsons-table-2.qza \
--m-metadata-file parkinsons_metadata_sorted_grouped.tsv \
--p-where "[Disease]='Control'" \
--o-filtered-table control-filtered-table.qza

## Visualize Filtered tables
# 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/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv

# Visualize control filtered table
qiime feature-table summarize \
--i-table control-filtered-table.qza \
--o-visualization control-filtered-table.qzv \
--m-sample-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv

## Determining Sampling Depth
​​# Look at parkinsons-table.qzv, control-filtered-table.qzv, and PD-filtered-table.qzv files 

## Alpha-rarefaction
# Alpha-rarefaction for non-filtered 
qiime diversity alpha-rarefaction \
--i-table parkinsons-table.qza \
--i-phylogeny rooted-tree.qza \
--p-max-depth 10232 \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--o-visualization alpha-rarefaction.qzv
 
# Alpha-rarefaction for control-filtered
qiime diversity alpha-rarefaction \
--i-table control-filtered-table.qza \
--i-phylogeny rooted-tree.qza \
--p-max-depth 10232 \
--m-metadata-file parkinsons_metadata_sorted_grouped.tsv \
--o-visualization control-filtered-alpha-rarefaction.qzv

# Alpha-rarefaction for pd-filtered
qiime diversity alpha-rarefaction \
--i-table pd-filtered-table.qza \
--i-phylogeny rooted-tree.qza \
--p-max-depth 10232 \
--m-metadata-file parkinsons_metadata_sorted_grouped.tsv \
--o-visualization pd-filtered-alpha-rarefaction.qzv

## Diversity Metrics for PD
# Calculate alpha- and beta- diversity metrics
qiime diversity core-metrics-phylogenetic \
--i-phylogeny rooted-tree.qza \
--i-table pd-filtered-table.qza \
--p-sampling-depth 10232 \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--output-dir pd-core-metrics-results

# Calculate alpha-group-significance - Faith’s & Evenness
qiime diversity alpha-group-significance \
--i-alpha-diversity pd-core-metrics-results/faith_pd_vector.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization pd-core-metrics-results/pd-faith-pd-group-significance.qzv

qiime diversity alpha-group-significance \  
--i-alpha-diversity pd-core-metrics-results/evenness_vector.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization pd-core-metrics-results/pd-evenness-group-significance.qzv

# Principal Coordinates Analysis (PCoA) - Unweighted Unifrac & Bray Curtis 
qiime emperor plot \
--i-pcoa pd-core-metrics-results/unweighted_unifrac_pcoa_results.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization pd-core-metrics-results/pd-unweighted-unifrac-emperor.qzv

qiime emperor plot \
--i-pcoa pd-core-metrics-results/bray_curtis_pcoa_results.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization pd-core-metrics-results/pd-bray-curtis-emperor.qzv

## Diversity Metrics for Control
qiime diversity core-metrics-phylogenetic \
--i-phylogeny rooted-tree.qza \
--i-table control-filtered-table.qza \
--p-sampling-depth 10232 \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--output-dir control-core-metrics-results-2


# Calculate alpha-group-significance - Faith’s & Evenness
qiime diversity alpha-group-significance \
--i-alpha-diversity control-core-metrics-results-2/faith_pd_vector.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization control-core-metrics-results-2/control-faith-pd-group-significance-2.qzv


qiime diversity alpha-group-significance \
--i-alpha-diversity control-core-metrics-results-2/evenness_vector.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization control-core-metrics-results-2/control-evenness-group-significance-2.qzv
  
# Principal Coordinates Analysis (PCoA) - Unweighted Unifrac & Bray Curtis
qiime emperor plot \
--i-pcoa control-core-metrics-results-2/unweighted_unifrac_pcoa_results.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization control-core-metrics-results-2/control-unweighted-unifrac-emperor-2.qzv

qiime emperor plot \
--i-pcoa control-core-metrics-results-2/bray_curtis_pcoa_results.qza \
--m-metadata-file /data/parkinsons_metadata/parkinsons_metadata_sorted_grouped.tsv \
--o-visualization control-core-metrics-results-2/control-bray-curtis-emperor-2.qzv

## Diversity Metrics for unfiltered
# Calculate alpha- and beta- diversity metrics
qiime diversity core-metrics-phylogenetic \
--i-phylogeny rooted-tree.qza \
--i-table parkinsons-table.qza \
--p-sampling-depth 10232 \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--output-dir unfiltered-core-metrics-results

# Calculate alpha-group-significance - Faith’s & Evenness
qiime diversity alpha-group-significance \
--i-alpha-diversity pd-core-metrics-results/faith_pd_vector.qza \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--o-visualization unfiltered-core-metrics-results/unfiltered-faith-pd-group-significance.qzv

qiime diversity alpha-group-significance \  
--i-alpha-diversity pd-core-metrics-results/evenness_vector.qza \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--o-visualization unfiltered-core-metrics-results/unfiltered-evenness-group-significance.qzv

# Principal Coordinates Analysis (PCoA) - Unweighted Unifrac & Bray Curtis 
qiime emperor plot \
--i-pcoa unfiltered-core-metrics-results/unweighted_unifrac_pcoa_results.qza \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--o-visualization unfiltered-core-metrics-results/unfiltered-unweighted-unifrac-emperor.qzv

qiime emperor plot \
--i-pcoa unfiltered-core-metrics-results/bray_curtis_pcoa_results.qza \
--m-metadata-file /mnt/datasets/project_2/parkinsons/parkinsons_metadata.txt \
--o-visualization unfiltered-core-metrics-results/unfiltered-bray-curtis-emperor.qzv