# Import via manifest into qiime and demultiplex
qiime tools import \
  --type "SampleData[SequencesWithQuality]" \
  --input-format SingleEndFastqManifestPhred33V2 \
  --input-path cleaned_manifest.tsv \
  --output-path demux_seqs.qza

# Visualize demultiplexed sequences
qiime demux summarize \
  --i-data demux_seqs.qza \
  --o-visualization demux.qzv

# Denoising and clustering using dada2
qiime dada2 denoise-single \
  --i-demultiplexed-seqs demux_seqs.qza \
  --p-trim-left 0 \
  --p-trunc-len 240 \
  --o-representative-sequences rep-seqs.qza \
  --o-table table.qza \
  --o-denoising-stats stats.qza

# Visualize table.qza
qiime feature-table summarize \
  --i-table table.qza \
  --o-visualization table.qzv \
  --m-sample-metadata-file meta.tsv

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

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

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

# Filter out chloroplast/mitochondria data
qiime taxa filter-table \
  --i-table table.qza \
  --i-taxonomy taxonomy.qza \
  --p-exclude mitochondria,chloroplast \
  --o-filtered-table filtered_table.qza

 # Visualize filtered table
 qiime feature-table summarize \
  --i-table filtered_table.qza \
  --o-visualization filtered_table.qzv \
  --m-sample-metadata-file meta.tsv 

 # Generate a phylogenetic tree
 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 curve
qiime diversity alpha-rarefaction \
  --i-table table.qza \
  --i-phylogeny rooted-tree.qza \
  --p-max-depth 13000 \
  --m-metadata-file meta.tsv \
  --o-visualization alpha-rarefaction.qzv

# Core metrics for diversity analyses
qiime diversity core-metrics-phylogenetic \
  --i-phylogeny rooted-tree.qza \
  --i-table filtered_table.qza \
  --p-sampling-depth 1704 \
  --m-metadata-file meta.tsv \
  --output-dir core-metrics-results

# Export qiime artifacts for downstream analysis in R

# Table
qiime tools export \
--input-path ../filtered_table.qza \
--output-path filtered_table 

# Convert biom to txt
biom convert 
-i feature-table.biom 
--to-tsv -o feature-table.txt

# Tree
qiime tools export \
--input-path ../rooted-tree.qza \
--output-path rooted_tree

# Taxonomy
qiime tools export \
--input-path ../taxonomy.qza \
--output-path taxonomy


