directories and sh

qiimePreprocessing/qiime2.sh

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+#! usr/bin/bash
+
+# download env config
+curl -sL \
+  "https://data.qiime2.org/distro/core/qiime2-2020.8-py36-osx-conda.yml" > \
+  "qiime2.yml"
+
+# create conda env
+conda env create -n qiime2 --file qiime2.yml
+
+# remove env file
+rm qiime2.yml
+
+# activate conda env
+conda activate qiime2
+
+# import data
+qiime tools import \
+  --type 'SampleData[PairedEndSequencesWithQuality]' \
+  --input-path ./manifest1.tsv \
+  --input-format PairedEndFastqManifestPhred33V2 \
+  --output-path demux.qza
+
+# summariza data and create a visualization for the quality of the reads
+qiime demux summarize --i-data demux.qza \
+--o-visualization demux.qzv
+
+# trimm primers off
+qiime cutadapt trim-paired --i-demultiplexed-sequences demux.qza \
+--p-front-f CCTACGGGNGGCWGCAG \
+--p-front-r GACTACHVGGGTATCTAATCC \
+--p-discard-untrimmed \
+--o-trimmed-sequences demux-trimmed.qza \
+--verbose
+
+# denoise with DADA2
+qiime dada2 denoise-paired --i-demultiplexed-seqs demux-trimmed.qza \
+--p-trunc-len-f 280 \
+--p-trunc-len-r 170 \
+--p-n-threads 4 \
+--output-dir DADA2_denoising_output \
+--verbose
+
+# summarize outputs
+# table
+qiime feature-table summarize \
+--i-table /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/table.qza \
+--o-visualization table \
+--m-sample-metadata-file metadata.tsv
+
+# rep_seqs
+qiime feature-table tabulate-seqs \
+--i-data /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/representative_sequences.qza \
+--o-visualization rep_seqs
+
+# denoising stats
+qiime metadata tabulate \
+--m-input-file /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/denoising_stats.qza \
+--o-visualization denoising_stats
+
+# build phylogenetic tree
+# align with mafft
+qiime alignment mafft  \
+--i-sequences /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/representative_sequences.qza \
+--o-alignment aligned_rep_seqs.qza
+
+# mask alignment
+qiime alignment mask --i-alignment aligned_rep_seqs.qza \
+--o-masked-alignment masked_aligned_rep_seqs.qza
+
+# build an unrooted tree
+qiime phylogeny fasttree \
+--i-alignment masked_aligned_rep_seqs.qza --o-tree unrooted_tree
+
+# root the tree
+qiime phylogeny midpoint-root \
+--i-tree unrooted_tree.qza --o-rooted-tree rooted_tree
+
+# diversity analyses
+qiime diversity core-metrics-phylogenetic \
+--i-phylogeny rooted_tree.qza \
+--i-table /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/table.qza \
+--p-sampling-depth 19000 --m-metadata-file metadata.tsv \
+--output-dir core_metrics_results
+
+# create alpha div visualizations
+qiime diversity alpha-group-significance \
+--i-alpha-diversity core_metrics_results/faith_pd_vector.qza \
+--m-metadata-file metadata.tsv \
+--o-visualization core_metrics_results/faith_pd_group_significance
+
+qiime diversity alpha-group-significance \
+--i-alpha-diversity core_metrics_results/evenness_vector.qza \
+--m-metadata-file metadata.tsv \
+ --o-visualization core_metrics_results/evenness_group_significance
+
+qiime diversity alpha-group-significance \
+--i-alpha-diversity core_metrics_results/shannon_vector.qza --m-metadata-file metadata.tsv \
+--o-visualization core_metrics_results/shannon_group_significance
+
+# alpha rarefaction plots
+qiime diversity alpha-rarefaction \
+--i-table core_metrics_results/rarefied_table.qza \
+--p-max-depth 30000 --m-metadata-file metadata.tsv \
+--p-steps 25 --o-visualization alpha_rarefaction.qzv
+
+# train classifier
+# download silva files https://www.arb-silva.de/download/archive/qiime/
+
+# import silva files
+qiime tools import --type 'FeatureData[Sequence]' \
+--input-path /Users/natmurad/Documents/Buck/Dipa16S/SILVA_132_QIIME_release/rep_set/rep_set_16S_only/99/silva_132_99_16S.fna \
+--output-path silva132_99
+
+qiime tools import --type 'FeatureData[Taxonomy]' \
+--input-format HeaderlessTSVTaxonomyFormat \
+--input-path /Users/natmurad/Documents/Buck/Dipa16S/SILVA_132_QIIME_release/taxonomy/16S_only/99/taxonomy_7_levels.txt \
+--output-path silva132_99_ref_taxonomy
+
+# extract ref reads
+qiime feature-classifier extract-reads --i-sequences silva132_99.qza \
+--p-f-primer GTGCCAGCMGCCCGCGGTAA --p-r-primer GGACTACHVGGGTWTCTAAT \
+--p-trunc-len 300 --p-n-jobs 5 --o-reads ref_seqs
+
+# train classifier
+qiime feature-classifier fit-classifier-naive-bayes \
+--i-reference-reads ref_seqs.qza \
+--i-reference-taxonomy silva132_99_ref_taxonomy.qza \
+--o-classifier classifier.qza
+
+# assign taxonomy
+qiime feature-classifier classify-sklearn \
+--i-classifier /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/silva-138-99-nb-classifier.qza \
+--i-reads /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/representative_sequences.qza \
+--o-classification taxonomy.qza
+
+# create visualization
+qiime metadata tabulate --m-input-file taxonomy.qza \
+--o-visualization taxonomy
+
+# create barplots
+qiime taxa barplot --i-table core_metrics_results/rarefied_table.qza \
+--i-taxonomy taxonomy.qza \
+--m-metadata-file metadata.tsv --o-visualization taxa_bar_plots.qzv
+
+# export to tsv
+qiime tools export \
+--input-path /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/DADA2_denoising_output/table.qza \
+--output-path export/table
+
+biom convert -i /Users/natmurad/Documents/Buck/Dipa16S/emp-paired-end-sequences/export/table/feature-table.biom \
+-o table.tsv --to-ts