#Attempt 4 at 16S rRNA metagonomic samples #Erinne Stirling - 07/09/2018 #################### START #################### #In terminal source activate qiime2-2018.6 source tab-qiime #Change to working directory cd ~/Documents/BioInf/Divstats/16s_workspace/ #################### FILES #################### #Validate metadata file used Keemei: cloud-based validation of tabular bioinformatics file formats in Google Sheets. Rideout JR, Chase JH, Bolyen E, Ackermann G, González A, Knight R, Caporaso JG. GigaScience. 2016;5:27. http://dx.doi.org/10.1186/s13742-016-0133-6 #Metadata file: ~/divstats-meta.tsv #Manifest file: /Documents/BioInf/Divstats/files/AGRF_CAGRF17715_BVF5M/manifest.txt #################### IMPORT #################### cd ~/Documents/BioInf/Divstats/files/AGRF_CAGRF17715_BVF5M qiime tools import \ --type 'SampleData[PairedEndSequencesWithQuality]' \ --input-path manifest.txt \ --source-format PairedEndFastqManifestPhred33 \ --output-path ~/Documents/BioInf/Divstats/16s_workspace/demux.qza cd ~/Documents/BioInf/Divstats/16s_workspace/ #Visualise the data qiime demux summarize \ --i-data demux.qza \ --o-visualization demux.qzv qiime tools view demux.qzv #Add metadata qiime metadata tabulate \ --m-input-file divstats-meta.tsv \ --o-visualization tabulated_meta.qzv qiime tools view tabulated_meta.qzv #################### DEBLUR #################### #Generate quality filter qiime quality-filter q-score \ --i-demux demux.qza \ --o-filtered-sequences demux-filtered.qza \ --o-filter-stats demux-filter-stats.qza #Trim data for quality qiime deblur denoise-16S \ --i-demultiplexed-seqs demux-filtered.qza \ --p-trim-length 250 \ --o-representative-sequences rep-seqs-deblur.qza \ --o-table table-deblur.qza \ --p-sample-stats \ --o-stats deblur-stats.qza #Check quality qiime metadata tabulate \ --m-input-file demux-filter-stats.qza \ --o-visualization demux-filter-stats.qzv qiime tools view demux-filter-stats.qzv qiime deblur visualize-stats \ --i-deblur-stats deblur-stats.qza \ --o-visualization deblur-stats.qzv qiime tools view deblur-stats.qzv #Rename files mv rep-seqs-deblur.qza rep-seqs.qza mv table-deblur.qza table.qza #################### ANALYSIS PREP #################### #Sequence number per sample qiime feature-table summarize \ --i-table table.qza \ --o-visualization table.qzv \ --m-sample-metadata-file divstats-meta.tsv qiime tools view table.qzv #------------------------------------------------------Sample depth = 5900 qiime feature-table tabulate-seqs \ --i-data rep-seqs.qza \ --o-visualization rep-seqs.qzv qiime tools view rep-seqs.qzv #################### TREES #################### #Alignment qiime alignment mafft \ --i-sequences rep-seqs.qza \ --o-alignment aligned-rep-seqs.qza #Mask the highly variable bits qiime alignment mask \ --i-alignment aligned-rep-seqs.qza \ --o-masked-alignment masked-aligned-rep-seqs.qza #Fasttree qiime phylogeny fasttree \ --i-alignment masked-aligned-rep-seqs.qza \ --o-tree unrooted-tree.qza #Root it qiime phylogeny midpoint-root \ --i-tree unrooted-tree.qza \ --o-rooted-tree rooted-tree.qza #################### DIVERSITY - ALPHA #################### qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table.qza \ --p-sampling-depth 5900 \ --m-metadata-file divstats-meta.tsv \ --output-dir core-metrics-results #***FAITH***# qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file divstats-meta.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime tools view core-metrics-results/faith-pd-group-significance.qzv #------------------------------------------------------Kruskall-Wallace #------------------------------------------------------H 15.97 #------------------------------------------------------P 0.0031 qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file divstats-meta.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv qiime tools view core-metrics-results/evenness-group-significance.qzv #------------------------------------------------------Kruskall-Wallace #------------------------------------------------------H 9.19 #------------------------------------------------------P 0.0564 qiime diversity alpha-correlation \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file divstats-meta.tsv \ --o-visualization core-metrics-results/faith-alpha-correlation.qzv qiime tools view core-metrics-results/faith-alpha-correlation.qzv #------------------------------------------------------Respiration #------------------------------------------------------Spearman #------------------------------------------------------Test-stat -0.6692 #------------------------------------------------------P 0.0003 #***SHANNON***# qiime diversity alpha \ --i-table table.qza \ --p-metric shannon \ --o-alpha-diversity shannon.qza qiime diversity alpha-group-significance \ --i-alpha-diversity shannon.qza \ --m-metadata-file divstats-meta.tsv \ --o-visualization shannon.qzv qiime tools view Shannon.qzv #################### DIVERSITY - BETA #################### qiime diversity beta-group-significance \ --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file divstats-meta.tsv \ --m-metadata-column Temp \ --o-visualization core-metrics-results/unweighted-unifrac-temp-significance.qzv \ --p-pairwise qiime tools view core-metrics-results/unweighted-unifrac-temp-significance.qzv #------------------------------------------------------PERMANOVA #------------------------------------------------------pseudo-F #------------------------------------------------------Test-stat 2.96621 #------------------------------------------------------P 0.001 #------------------------------------------------------PAIRWISE #------------------------------------------------------P<0.05 40.C-> control #------------------------------------------------------ 260 #------------------------------------------------------ 320 #------------------------------------------------------ 150.C-> control #------------------------------------------------------ 260 #------------------------------------------------------ 320 #------------------------------------------------------ 260.C-> 40 #------------------------------------------------------ 150 #------------------------------------------------------ 320.C-> control #------------------------------------------------------ 40 #------------------------------------------------------ 150 qiime diversity beta-group-significance \ --i-distance-matrix core-metrics-results/weighted_unifrac_distance_matrix.qza \ --m-metadata-file divstats-meta.tsv \ --m-metadata-column Temp \ --o-visualization core-metrics-results/weighted-unifrac-temp-significance.qzv \ --p-pairwise qiime tools view core-metrics-results/weighted-unifrac-temp-significance.qzv #################### TRAIN A CLASSIFIER #################### qiime tools import \ --type 'FeatureData[Sequence]' \ --input-path gg_13_8_otus/rep_set/99_otus.fasta \ --output-path 99_otus.qza qiime tools import \ --type 'FeatureData[Taxonomy]' \ --source-format HeaderlessTSVTaxonomyFormat \ --input-path gg_13_8_otus/taxonomy/99_otu_taxonomy.txt \ --output-path ref-taxonomy.qza qiime feature-classifier extract-reads \ --i-sequences 99_otus.qza \ --p-f-primer AGAGTTTGATCMTGGCTCAG \ --p-r-primer GWATTACCGCGGCKGCTG \ --p-trunc-len 250 \ --o-reads ref-seqs.qza qiime feature-classifier fit-classifier-naive-bayes \ --i-reference-reads ref-seqs.qza \ --i-reference-taxonomy ref-taxonomy.qza \ --o-classifier classifier.qza #################### TAXONOMY BACTERIA#################### qiime feature-classifier classify-sklearn \ --i-classifier classifier.qza \ --i-reads rep-seqs.qza \ --o-classification taxonomy.qza qiime metadata tabulate \ --m-input-file taxonomy.qza \ --o-visualization taxonomy.qzv qiime tools view taxonomy.qzv qiime taxa barplot \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --m-metadata-file divstats-meta.tsv \ --o-visualization taxa-bar-plots.qzv qiime tools view taxa-bar-plots.qzv #################### TAXONOMY ARCHAEA#################### qiime feature-classifier classify-sklearn \ --i-classifier silva-132-99-nb-classifier.qza \ --i-reads rep-seqs.qza \ --o-classification taxonomy_archaea.qza qiime metadata tabulate \ --m-input-file taxonomy_archaea.qza \ --o-visualization taxonomy_archaea.qzv #################### q2-composition #################### qiime composition add-pseudocount \ --i-table table.qza \ --o-composition-table comp-table.qza qiime composition ancom \ --i-table comp-table.qza \ --m-metadata-file divstats-meta.tsv \ --m-metadata-column Temp \ --o-visualization ancom-Temp.qzv ### qiime taxa collapse \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --p-level 3 \ --o-collapsed-table table-l3.qza qiime composition add-pseudocount \ --i-table table-l2.qza \ --o-composition-table comp-table-l3.qza qiime composition ancom \ --i-table comp-table-l3.qza \ --m-metadata-file divstats-meta.tsv \ --m-metadata-column Temp \ --o-visualization ancom-Temp-l3.qzv ### qiime taxa collapse \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --p-level 6 \ --o-collapsed-table table-l6.qza qiime composition add-pseudocount \ --i-table table-l6.qza \ --o-composition-table comp-table-l6.qza qiime composition ancom \ --i-table comp-table-l6.qza \ --m-metadata-file divstats-meta.tsv \ --m-metadata-column Temp \ --o-visualization ancom-Temp-l6.qzv