Qiime2-2017.12

#Generate OTU table (feature table), Assign Taxonomy (SILVA full length) and generate the phylogenetic tree

#Import sequence files to Qiime2
#manifest phred33

#create a manifest file
#rename file names as in the manifest 

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path pe-33-manifest --output-path paired-end-demux.qza --source-format PairedEndFastqManifestPhred33

#quality check

qiime demux summarize \
  --i-data paired-end-demux.qza \
  --o-visualization demux-summary-1.qzv

#The sequences are sorted inline from eurofins, sequences sorted by barcode then trimmed. 

  
#quality_filter 
  
 qiime quality-filter q-score \
 --i-demux paired-end-demux.qza \
 --o-filtered-sequences demux-filtered.qza \
 --o-filter-stats demux-filter-stats.qza


#denoise_after_filtering 
 
 qiime deblur denoise-16S \
  --i-demultiplexed-seqs demux-filtered.qza \
  --p-trim-length 230 \
  --o-representative-sequences rep-seqs-deblur.qza \
  --o-table table-deblur.qza \
  --p-sample-stats \
  --o-stats deblur-stats.qza


mv rep-seqs-deblur.qza rep-seqs.qza
mv table-deblur.qza table.qza


#create qiime visualizations

qiime metadata tabulate \
  --m-input-file demux-filter-stats.qza \
  --o-visualization demux-filter-stats.qzv

qiime deblur visualize-stats \
  --i-deblur-stats deblur-stats.qza \
  --o-visualization deblur-stats.qzv






#create a metadata file on google sheets and save as (.tsv)
#feature table and feature summary 

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


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


#Generate a tree for phylogenetic diversity analyses

qiime alignment mafft \
  --i-sequences rep-seqs.qza \
  --o-alignment aligned-rep-seqs.qza
  

#mask or filter

qiime alignment mask \
  --i-alignment aligned-rep-seqs.qza \
  --o-masked-alignment masked-aligned-rep-seqs.qza
  
#create a phylogenetic tree 

qiime phylogeny fasttree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --o-tree unrooted-tree.qza


#taxonomy_silva_full_length 

qiime feature-classifier classify-sklearn \
  --i-classifier silva-119-99-nb-classifier.qza \
  --i-reads rep-seqs.qza \
  --o-classification taxonomy.qza


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


#bar_plot

qiime taxa barplot \
  --i-table table.qza \
  --i-taxonomy taxonomy.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization taxa-bar-plots.qzv


#export to biom hdf5


qiime tools export \
  table.qza \
  --output-dir exported-feature-table



#export the following files to be used in R-phyloseq:
feature table in biom format
taxonomy (export as tsv)
re_seq_file.fasta
tree.nwk
metadata.tsv

#append taxonomy classification to feature table and convert to biom format

biom add-metadata -i feature-table.biom -o table.w_omd.biom --observation-metadata-fp taxonomy.tsv --observation-header OTUID,taxonomy --sc-separated taxonomy