# Step1 import data
 import=${results}/01.import-to-qiime2
 mkdir ${import}
 qiime tools import \
    --type 'SampleData[SequencesWithQuality]' \
    --input-path ${import}/manifest.tsv \
    --output-path ${import}/single-end-demux.qza \
    --input-format SingleEndFastqManifestPhred33V2

 qiime demux summarize \
    --i-data ${import}/single-end-demux.qza \
    --o-visualization ${import}/single-end-demux.qzv

# step2 quality control

qc=${results}/02.quality-filter
mkdir ${qc}

qiime quality-filter q-score \
    --i-demux ${import}/single-end-demux.qza \
    --o-filtered-sequences ${qc}/single-end-demux-filtered.qza \
    --o-filter-stats ${qc}/single-end-demux-filtered-stats.qza
qiime  metadata tabulate \
    --m-input-file ${qc}/single-end-demux-filtered-stats.qza \
    --o-visualization ${qc}/single-end-demux-filtered-stats.qzv


# Step3 DADA2 denoising 

dada2=${results}/03.dada2-denoise
mkdir ${dada2}

qiime dada2  denoise-single \
    --i-demultiplexed-seqs ${qc}/single-end-demux-filtered.qza \
    --p-trunc-len 250 \
    --p-trim-left 35 \
    --p-n-threads 60 \
    --o-table ${dada2}/dada2-table.qza \
    --o-representative-sequences ${dada2}/dada2-rep-seqs.qza \
    --o-denoising-stats ${dada2}/dada2-stats.qza 

qiime feature-table tabulate-seqs \
    --i-data ${dada2}/dada2-rep-seqs.qza \
    --o-visualization ${dada2}/dada2-rep-seqs.qzv 

# Step 4 Taxonomic annot

taxonomy=${results}/04.taxonomy-annot
mkdir ${taxonomy}
database=${taxonomy}/01.database
annotDir=${taxonomy}/02.annotation
mkdir -p ${database} ${annotDir}

qiime feature-classifier classify-sklearn \
    --p-n-jobs 128 \
    --i-classifier ${database}/silva-138-99-nb-classifier-full.qza \
    --i-reads ${dada2}/dada2-rep-seqs.qza \
    --o-classification ${annotDir}/dada2-rep-seqs-taxonomy.qza


qiime metadata tabulate \
    --m-input-file ${annotDir}/dada2-rep-seqs-taxonomy.qza \
    --o-visualization ${annotDir}/dada2-rep-seqs-taxonomy.qzv


# Step 5 ASV filter

asvFilter=${results}/05.asv-filter
mkdir ${asvFilter}

# remove doubletons
 qiime feature-table filter-features \
    --i-table ${dada2}/dada2-table.qza \
    --p-min-frequency 2 \
    --o-filtered-table ${asvFilter}/dada2-frequency-filtered-table.qza

 qiime feature-table summarize \
    --i-table ${asvFilter}/dada2-frequency-filtered-table.qza \
    --o-visualization ${asvFilter}/dada2-frequency-filtered-table.qzv \
    --m-sample-metadata-file ${metadata}



# remove ASVs less than two samples

 qiime feature-table filter-features \
    --i-table ${asvFilter}/dada2-frequency-filtered-table.qza \
    --p-min-samples 2 \
    --o-filtered-table ${asvFilter}/dada2-contingency-filtered-table.qza

 qiime feature-table summarize \
    --i-table ${asvFilter}/dada2-contingency-filtered-table.qza \
    --o-visualization ${asvFilter}/dada2-contingency-filtered-table.qzv \
    --m-sample-metadata-file ${metadata}

# remove choloroplast and mitochondrion

qiime taxa filter-table \
    --i-table ${asvFilter}/dada2-contingency-filtered-table.qza \
    --i-taxonomy ${annotDir}/dada2-rep-seqs-taxonomy.qza \
    --p-exclude mitochondria,chloroplast \
    --o-filtered-table ${asvFilter}/table-no-mitochondria-no-chloroplast-final.qza

qiime feature-table summarize \
    --i-table ${asvFilter}/table-no-mitochondria-no-chloroplast-final.qza \
    --o-visualization ${asvFilter}/table-no-mitochondria-no-chloroplast-final.qzv \
    --m-sample-metadata-file ${metadata}



## Step 6 Resampling

resample=${results}/06.asv-resample
mkdir ${resample}

qiime feature-table rarefy \
    --i-table ${asvFilter}/table-no-mitochondria-no-chloroplast-final.qza \
    --p-sampling-depth 18900 \
    --o-rarefied-table ${resample}/table-no-mitochondria-no-chloroplast-final-resampled.qza

qiime feature-table summarize \
    --i-table ${resample}/table-no-mitochondria-no-chloroplast-final-resampled.qza \
    --o-visualization ${resample}/table-no-mitochondria-no-chloroplast-final-resampled.qzv \
    --m-sample-metadata-file ${metadata}

# Step 7 Taxonomic composition

taxbarplot=${results}/07.taxon-barplot
mkdir ${taxbarplot}

 qiime taxa barplot \
    --i-table ${resample}/table-no-mitochondria-no-chloroplast-final-resampled.qza \
    --m-metadata-file ${metadata} \
    --i-taxonomy ${annotDir}/dada2-rep-seqs-taxonomy.qza \
    --o-visualization ${taxbarplot}/table-no-mitochondria-no-chloroplast-final-resampled-barplot.qzv

#Step 8 Export ASV table

featrueTable=${results}/08.feature-table
mkdir -p ${featrueTable}

qiime tools export \
    --input-path ${resample}/table-no-mitochondria-no-chloroplast-final-resampled.qza \
    --output-path ${featrueTable}

qiime biom convert \
    -i ${featrueTable}/feature-table.biom \
    -o ${featrueTable}/feature-table.tsv \
    --to-tsv \
    --table-type 'OTU table'