#Project title: Larvae of deep-sea marine invertebrates harbor low-diversity bacterial communities
#Project authors: Tyler J. Carrier, Stace E. Beaulieu, Susan W. Mills, Lauren S. Mullineaux, and Adam M. Reitzel

#What: Computational pipeline for the V3/V4 region of the 16S rRNA gene (i.e., phylogenetic marker for bacteria), QIIME2 (v 2019.1), SILVA (v 132), and Amplicon Sequence Variants (ASVs)



### STEP 1: IMPORT RAW FILES INTO QIIME2 ###
qiime tools import \
  --type 'SampleData[PairedEndSequencesWithQuality]' \
  --input-path 
  --input-format CasavaOneEightSingleLanePerSampleDirFmt \
  --output-path 



### STEP 2: TRIM PRIMERS OFF RAW READS #
qiime cutadapt trim-paired \
  --i-demultiplexed-sequences 
  --p-front-f CCTACGGGNGGCWGCAG \
  --p-front-r GACTACHVGGGTATCTAATCC \
  --p-error-rate 0 \
  --o-trimmed-sequences 



### STEP 3: JOIN/MERGE PAIRED-ENDS ###
qiime vsearch join-pairs \
  --i-demultiplexed-seqs 
  --o-joined-sequences 
  --p-minovlen 20 \
  --p-maxdiffs 10 \
  --p-minmergelen 350 \
  --p-maxmergelen 550 \
  --p-allowmergestagger \
  --p-truncqual 10 \
  --p-minlen 100 \
  --p-qmax 41 \
  --p-qmaxout 41



### STEP 4: QUALITY CONTROL PAIRED-END READS ###
qiime quality-filter q-score-joined \
  --i-demux 
  --o-filtered-sequences 
  --o-filter-stats 
  --p-quality-window 5 \
  --p-min-quality 25 



### STEP 5: REIMPORT FILTERED FILES WITH MODIFIED NAMES ###
	#NOTE: THIS IS OPTIONAL
qiime tools export \
  --input-path 
  --output-path 

qiime tools import \
  --type 'SampleData[SequencesWithQuality]' \
  --input-path 
  --output-path 
  --input-format SingleEndFastqManifestPhred33



### STEP 6: DENOISE PROCESSED READS ###
qiime deblur denoise-16S \
  --i-demultiplexed-seqs 
  --p-trim-length 400 \
  --o-representative-sequences
  --o-table
  --p-sample-stats \
  --o-stats



### STEP 7: CREATE PHYLOGENY ###
#ALIGNMENT OF REPRESENTATIVE SEQUENCES
qiime alignment mafft \
  --i-sequences 
  --o-alignment 

#MASK HIGHLY VARIABLE NOISY POSITIONS IN ALIGNMENT
qiime alignment mask \
  --i-alignment 
  --o-masked-alignment

#CREATE PHYLOGENY WITH FASTTREE
qiime phylogeny fasttree \
  --i-alignment 
  --o-tree 

#ROOT PHYLOGENY AT MIDPOINT
qiime phylogeny midpoint-root \
  --i-tree 
  --o-rooted-tree 



### STEP 8: ASSIGN TAXONOMY WITH TRAINED CLASSIFIER ###
qiime feature-classifier classify-sklearn \
  --i-classifier silva-132-99-classifier.qza \
  --i-reads 
  --o-classification 
  --p-confidence 
  --p-read-orientation same



### STEP 9: FILTER ARCHAEA ###
qiime taxa filter-table \
  --i-table 
  --i-taxonomy 
  --p-exclude Archaea \
  --p-mode contains \
  --o-filtered-table 



### STEP 10: FILTER FEATURES FROM DNA SAMPLES ###
#IDENTIFY FEATURES IN DNA KIT BLANKS
qiime tools export \
  --input-path 
  --output-path 

biom convert \
  -i 
  -o 
  --to-tsv

#FILTER FEATURES FOUND IN DNA KIT BLANKS
qiime feature-table filter-features \
  --i-table 
  --m-metadata-file 
  --o-filtered-table 



### STEP 11: RAREIFY TABLE ###
qiime feature-table rarefy \
  --i-table
  --p-sampling-depth 392 \
  --p-with-replacement \
  --o-rarefied-table 



# NOTE: ALL STEPS ABOVE ARE PART OF PRE-PROCESSING. BELOW IS TAILORED TO THE DATASET AND APPROACH OF INTEREST AND, HERE, IS SPECIFIC TO THIS DATASET #

### FIGURE 2: Alpha rarefaction curve for deep-sea larvae ###
qiime diversity alpha-rarefaction \
  --i-table 
  --p-max-depth 392 \
  --i-phylogeny 
  --m-metadata-file 
  --output-dir 



### FIGURE 3 and S1: Alpha diversity for deep-sea larvae ###
#alpha-diversity, faith
qiime diversity alpha-phylogenetic \
  --i-table 
  --i-phylogeny 
  --p-metric faith_pd \
  --output-dir 

#alpha-diversity, observedOTUs
qiime diversity alpha \
  --i-table
  --p-metric observed_otus \
  --output-dir

#alpha-diversity, McintoshE
qiime diversity alpha \
  --i-table 
  --p-metric mcintosh_e \
  --output-dir 

#alpha-diversity, McintoshD
qiime diversity alpha \
  --i-table 
  --p-metric mcintosh_d \
  --output-dir 



### FIGURE 4: Relatedness of the bacterial communities of deep-sea larvae ###
#beta-diversity, unweighted UniFrac
qiime diversity beta-phylogenetic \
  --i-table 
  --i-phylogeny 
  --p-metric unweighted_unifrac \
  --p-variance-adjusted \
  --o-distance-matrix 

qiime diversity pcoa \
  --i-distance-matrix 
  --o-pcoa 

qiime tools export \
  --input-path 
  --output-path 

# export "ordination.txt" and change all '-' in sample labels to '.' and then make q1-mapping file that cooresponds with q2-metadata file. Import both q1-compatible files #

# in QIIME1 #
make_2d_plots.py \
  -i 
  -o 
  -m 
  -b 
  --ellipsoid_opacity 
  --scree

qiime diversity beta-group-significance \
  --i-distance-matrix 
  --m-metadata-file 
  --m-metadata-column 
  --p-method permanova \
  --p-pairwise \
  --o-visualization 

#microbial dendrograms
qiime diversity beta-rarefaction \
  --i-table 
  --p-metric unweighted_unifrac \
  --p-clustering-method upgma \
  --m-metadata-file 
  --p-sampling-depth 392 \
  --i-phylogeny 
  --o-visualization


#beta-diversity, weighted UniFrac
qiime diversity beta-phylogenetic \
  --i-table 
  --i-phylogeny 
  --p-metric weighted_unifrac \
  --p-variance-adjusted \
  --o-distance-matrix 

qiime diversity pcoa \
  --i-distance-matrix 
  --o-pcoa 

qiime tools export \
  --input-path 
  --output-path 

# export "ordination.txt" and change all '-' in sample labels to '.' and then make q1-mapping file that cooresponds with q2-metadata file. Import both q1-compatible files # 

# in q1 #
make_2d_plots.py \
  -i 
  -o 
  -m 
  -b 
  --ellipsoid_opacity 
  --scree

qiime diversity beta-group-significance \
  --i-distance-matrix 
  --m-metadata-file 
  --m-metadata-column 
  --p-method permanova \
  --p-pairwise \
  --o-visualization 

#microbial dendrograms
qiime diversity beta-rarefaction \
  --i-table 
  --p-metric weighted_unifrac \
  --p-clustering-method upgma \
  --m-metadata-file 
  --p-sampling-depth 392 \
  --i-phylogeny 
  --o-visualization 



### Figure 5: Bacterial taxa for deep-sea larvae ###
qiime taxa barplot \
  --i-table 
  --i-taxonomy 
  --m-metadata-file
  --output-dir