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script-qiime2-v3
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#!/bin/bash
#Script de shell para el uso de QIIME2 con secuencias propias
#SCRIPT para análisis de secuencias sin tener en cuenta los cultivos iniciadores y todas las horas muestreadas de la fermentación
#IMPORT THE SEQUENCING DATA TO A QIIME2 ARTIFACT (QZA)
qiime tools import \
--type 'SampleData[PairedEndSequencesWithQuality]' \
--input-path reads_16s \
--input-format CasavaOneEightSingleLanePerSampleDirFmt \
--output-path demux-paired-end-16s.qza
qiime tools import \
--type 'SampleData[PairedEndSequencesWithQuality]' \
--input-path reads_its \
--input-format CasavaOneEightSingleLanePerSampleDirFmt \
--output-path demux-paired-end-its.qza
#ANALYZE THE QUALITY OF THE DATA WITH VISUALIZATIONS (QZV)
qiime demux summarize \
--i-data demux-paired-end-16s.qza \
--o-visualization demux-16s.qzv
qiime demux summarize \
--i-data demux-paired-end-its.qza \
--o-visualization demux-its.qzv
#DENOISE THE DATA WITH DADA2
qiime dada2 denoise-paired \
--i-demultiplexed-seqs demux-paired-end-16s.qza \
--p-trunc-len-f 249 \
--p-trunc-len-r 0 \
--p-trim-left-f 13 \
--p-trim-left-r 6 \
--o-table table-16s.qza \
--o-representative-sequences rep-seqs-16s.qza \
--o-denoising-stats denoising-stats-16s.qza
qiime dada2 denoise-paired \
--i-demultiplexed-seqs demux-paired-end-its.qza \
--p-trunc-len-f 249 \
--p-trunc-len-r 224 \
--p-trim-left-f 13 \
--p-trim-left-r 8 \
--o-table table-its.qza \
--o-representative-sequences rep-seqs-its.qza \
--o-denoising-stats denoising-stats-its.qza
#TAXONOMIC ANALYSIS
SILVA 16S 515/806:
qiime feature-classifier classify-sklearn \
--i-classifier silva-138-99-515-806-nb-classifier.qza \
--i-reads rep-seqs-16s.qza \
--o-classification silva-138-99-tax-515-806.qza
qiime metadata tabulate \
--m-input-file silva-138-99-tax-515-806.qza \
--o-visualization taxonomy-16s.qzv
#UNITE ITS:
#https://forum.qiime2.org/t/fungal-its-analysis-tutorial/7351
awk '/^>/ {print($0)}; /^[^>]/ {print(toupper($0))}' sh_refs_qiime_ver8_99_10.05.2021_dev.fasta | tr -d ' ' > sh_refs_qiime_ver8_99_10.05.2021_dev_uppercase.fasta
cd sh_qiime_release_10.05.2021/developer
qiime tools import \
--type ‘FeatureData[Sequence]’ \
--input-path sh_refs_qiime_ver8_99_10.05.2021_dev_uppercase.fasta \
--output-path unite-ver8-99-seqs.qza
qiime tools import \
--type ‘FeatureData[Taxonomy]’ \
--input-format HeaderlessTSVTaxonomyFormat \
--input-path sh_taxonomy_qiime_ver8_99_10.05.2021_dev.txt \
--output-path unite-ver8-99-tax.qza
qiime feature-classifier fit-classifier-naive-bayes \
--i-reference-reads unite-ver8-99-seqs.qza \
--i-reference-taxonomy unite-ver8-99-tax.qza \
--o-classifier unite-ver8-99-classifier-2021.qza
qiime feature-classifier classify-sklearn \
--i-classifier unite-ver8-99-classifier-2021.qza \
--i-reads rep-seqs-its.qza \
--o-classification unite-its-tax.qza
qiime metadata tabulate \
--m-input-file unite-its-tax.qza \
--o-visualization taxonomy-its.qzv
#REMOVE UNWANTED TAXA 16S (A POSTERIORI)
qiime taxa filter-table \
--i-table table-16s.qza \
--i-taxonomy silva-138-99-tax-515-806.qza \
--p-include p__ \
--p-exclude mitochondria,chloroplast \
--o-filtered-table table-with-no-mitochondria-chloroplast-16s.qza
qiime taxa filter-table \
--i-table table-with-no-mitochondria-chloroplast-16s.qza \
--i-taxonomy silva-138-99-tax-515-806.qza \
--p-exclude "k__Archaea" \
--o-filtered-table table-with-no-mitochondria-chloroplasts-archaea.qza
qiime taxa filter-table \
--i-table table-with-no-mitochondria-chloroplasts-archaea.qza \
--i-taxonomy silva-138-99-tax-515-806.qza \
--p-exclude "k__Eukaryota" \
--o-filtered-table final-filtered-table-16s.qza
#GENERATE SUMMARIES OF THE ARTIFACTS
qiime feature-table summarize \
--i-table final-filtered-table-16s.qza \
--o-visualization table-16s.qzv \
--m-sample-metadata-file sample-metadata-16s.tsv
qiime feature-table tabulate-seqs \
--i-data rep-seqs-16s.qza \
--o-visualization rep-seqs-16s.qzv
qiime metadata tabulate \
--m-input-file denoising-stats-16s.qza \
--o-visualization denoising-stats-16s.qzv
qiime feature-table summarize \
--i-table table-its.qza \
--o-visualization table-its.qzv \
--m-sample-metadata-file sample-metadata-its.tsv
qiime feature-table tabulate-seqs \
--i-data rep-seqs-its.qza \
--o-visualization rep-seqs-its.qzv
qiime metadata tabulate \
--m-input-file denoising-stats-its.qza \
--o-visualization denoising-stats-its.qzv
#GENERATE A TREE FOR PHYLOGENETIC DIVERSITY ANALYSES
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences rep-seqs-16s.qza \
--o-alignment aligned-rep-seqs-16s.qza \
--o-masked-alignment masked-aligned-rep-seqs-16s.qza \
--o-tree unrooted-tree-16s.qza \
--o-rooted-tree rooted-tree-16s.qza
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences rep-seqs-its.qza \
--o-alignment aligned-rep-seqs-its.qza \
--o-masked-alignment masked-aligned-rep-seqs-its.qza \
--o-tree unrooted-tree-its.qza \
--o-rooted-tree rooted-tree-its.qza
#ALPHA AND BETA DIVERSITY ANALYSES
#16S:
qiime diversity core-metrics-phylogenetic \
--i-phylogeny rooted-tree-16s.qza \
--i-table final-filtered-table-16s.qza \
--p-sampling-depth 4647 \
--m-metadata-file sample-metadata-16s.tsv \
--output-dir core-metrics-results-16s
qiime diversity alpha-group-significance \
--i-alpha-diversity core-metrics-results-16s/faith_pd_vector.qza \
--m-metadata-file sample-metadata-16s.tsv \
--o-visualization core-metrics-results-16s/faith-pd-group-significance.qzv
qiime diversity alpha-group-significance \
--i-alpha-diversity core-metrics-results-16s/evenness_vector.qza \
--m-metadata-file sample-metadata-16s.tsv \
--o-visualization core-metrics-results-16s/evenness-group-significance.qzv
qiime diversity beta-group-significance \
--i-distance-matrix core-metrics-results-16s/unweighted_unifrac_distance_matrix.qza \
--m-metadata-file sample-metadata-16s.tsv \
--m-metadata-column fermentation_type \
--o-visualization core-metrics-results-16s/unweighted-unifrac-fermentation-type-significance.qzv \
--p-pairwise
qiime diversity beta-group-significance \
--i-distance-matrix core-metrics-results-16s/unweighted_unifrac_distance_matrix.qza \
--m-metadata-file sample-metadata-16s.tsv \
--m-metadata-column barrel_site \
--o-visualization core-metrics-results-16s/unweighted-unifrac-barrel_site-group-significance.qzv \
--p-pairwise
qiime emperor plot \
--i-pcoa core-metrics-results-16s/unweighted_unifrac_pcoa_results.qza \
--m-metadata-file sample-metadata-16s.tsv \
--p-custom-axes fermentation_time \
--o-visualization core-metrics-results-16s/unweighted-unifrac-emperor-fermentation_time.qzv
qiime emperor plot \
--i-pcoa core-metrics-results-16s/bray_curtis_pcoa_results.qza \
--m-metadata-file sample-metadata-16s.tsv \
--p-custom-axes fermentation_time \
--o-visualization core-metrics-results-16s/bray-curtis-emperor-fermentation_time.qzv
#ITS:
qiime diversity core-metrics-phylogenetic \
--i-phylogeny rooted-tree-its.qza \
--i-table table-its.qza \
--p-sampling-depth 1052 \
--m-metadata-file sample-metadata-its.tsv \
--output-dir core-metrics-results-its
qiime diversity alpha-group-significance \
--i-alpha-diversity core-metrics-results-its/faith_pd_vector.qza \
--m-metadata-file sample-metadata-its.tsv \
--o-visualization core-metrics-results-its/faith-pd-group-significance.qzv
qiime diversity alpha-group-significance \
--i-alpha-diversity core-metrics-results-its/evenness_vector.qza \
--m-metadata-file sample-metadata-its.tsv \
--o-visualization core-metrics-results-its/evenness-group-significance.qzv
qiime diversity beta-group-significance \
--i-distance-matrix core-metrics-results-its/unweighted_unifrac_distance_matrix.qza \
--m-metadata-file sample-metadata-its.tsv \
--m-metadata-column fermentation_type \
--o-visualization core-metrics-results-its/unweighted-unifrac-fermentation-type-significance.qzv \
--p-pairwise
qiime diversity beta-group-significance \
--i-distance-matrix core-metrics-results-its/unweighted_unifrac_distance_matrix.qza \
--m-metadata-file sample-metadata-its.tsv \
--m-metadata-column barrel_site \
--o-visualization core-metrics-results-its/unweighted-unifrac-barrel_site-group-significance.qzv \
--p-pairwise
qiime emperor plot \
--i-pcoa core-metrics-results-its/unweighted_unifrac_pcoa_results.qza \
--m-metadata-file sample-metadata-its.tsv \
--p-custom-axes fermentation_time \
--o-visualization core-metrics-results-its/unweighted-unifrac-emperor-fermentation_time.qzv
qiime emperor plot \
--i-pcoa core-metrics-results-its/bray_curtis_pcoa_results.qza \
--m-metadata-file sample-metadata-its.tsv \
--p-custom-axes fermentation_time \
--o-visualization core-metrics-results-its/bray-curtis-emperor-fermentation_time.qzv
#ALPHA RAREFACTION PLOTTING
qiime diversity alpha-rarefaction \
--i-table final-filtered-table-16s.qza \
--i-phylogeny rooted-tree-16s.qza \
--p-max-depth 7829 \
--m-metadata-file sample-metadata-16s.tsv \
--o-visualization alpha-rarefaction-16s.qzv
qiime diversity alpha-rarefaction \
--i-table table-its.qza \
--i-phylogeny rooted-tree-its.qza \
--p-max-depth 5585 \
--m-metadata-file sample-metadata-its.tsv \
--o-visualization alpha-rarefaction-its.qzv
#TAXONOMY BARPLOTS:
qiime taxa barplot \
--i-table final-filtered-table-16s.qza \
--i-taxonomy silva-138-99-tax-515-806.qza \
--m-metadata-file sample-metadata-16s.tsv \
--o-visualization taxa-bar-plots-16s.qzv
qiime taxa barplot \
--i-table table-its.qza \
--i-taxonomy unite-its-tax.qza \
--m-metadata-file sample-metadata-its.tsv \
--o-visualization taxa-bar-plots-its.qzv