#Working feature table: table-phyla-no-mt-no-infrequent.qza #Working sequences: seqs-phyla-no-mt-no-infrequent.qzv #Generation of two feature tables stratified by age #2-3d (60m) and 5-6d (72hrs) are analysed separately to remove confounding qiime feature-table filter-samples \ --i-table table-phyla-no-mt-no-infrequent.qza \ --m-metadata-file sample-metadata.tsv \ --p-where "[group] IN ('controlA60', 'susceptible', '5xRes60', '10xRes60')"\ --o-filtered-table table-60mins.qza qiime feature-table summarize \ --i-table table-60mins.qza \ --o-visualization table-60mins.qzv \ --m-sample-metadata-file sample-metadata.tsv qiime feature-table filter-samples \ --i-table table-phyla-no-mt-no-infrequent.qza \ --m-metadata-file sample-metadata.tsv \ --p-where "[group] IN ('controlA72', '1xRes72', '5xRes72', '10xRes72')"\ --o-filtered-table table-72hrs.qza qiime feature-table summarize \ --i-table table-72hrs.qza \ --o-visualization table-72hrs.qzv \ --m-sample-metadata-file sample-metadata.tsv #Mosquitoes aged 2-3 days #Alpha diversity qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences seqs-phyla-no-mt-no-infrequent.qza \ --o-alignment aligned-seqs-phyla-no-mt-no-infrequent.qza \ --o-masked-alignment masked-aligned-seqs-phyla-no-mt-no-infrequent.qza \ --o-tree unrooted-tree.qza \ --o-rooted-tree rooted-tree.qza qiime diversity alpha-rarefaction \ --i-table table-phyla-no-mt-no-infrequent.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 2359 \ --m-metadata-file sample-metadata.tsv \ --o-visualization alpha-rarefaction_2359.qzv qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table-60mins.qza \ --p-sampling-depth 2359 \ --m-metadata-file sample-metadata.tsv \ --output-dir rarefied-core-metrics-results-60mins qiime diversity alpha-group-significance \ --i-alpha-diversity rarefied-core-metrics-results-60mins/shannon_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization rarefied-core-metrics-results-60mins/shannon_vector.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity rarefied-core-metrics-results-60mins/faith_pd_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization rarefied-core-metrics-results-60mins/faith_pd_vector.qzv #Beta diversity (using non-rarefied data) qiime diversity beta \ --i-table table-60mins.qza \ --p-metric braycurtis \ --o-distance-matrix braycurtis_distance_matrix.qza qiime diversity pcoa \ --i-distance-matrix braycurtis_distance_matrix.qza \ --o-pcoa braycurtis_pcoa.qza qiime emperor plot \ --i-pcoa braycurtis_pcoa.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization braycurtis_emperor.qzv qiime diversity beta-group-significance \ --i-distance-matrix braycurtis_distance_matrix.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column status \ --o-visualization braycurtis_distance_res_status.qzv \ --p-pairwise qiime diversity beta-phylogenetic \ --i-table table-60mins.qza \ --i-phylogeny rooted-tree.qza \ --p-metric weighted_normalized_unifrac \ --o-distance-matrix weighted_unifrac_distance_matrix.qza qiime diversity pcoa \ --i-distance-matrix weighted_unifrac_distance_matrix.qza \ --o-pcoa weighted_unifrac_pcoa.qza qiime emperor plot \ --i-pcoa weighted_unifrac_pcoa.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization weighted_unifrac_emperor.qzv qiime diversity beta-group-significance \ --i-distance-matrix weighted_unifrac_distance_matrix.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column status \ --o-visualization weighted_unifrac_distance_res_status.qzv \ --p-pairwise #pairways permanova with rarefied data for Bray-Curtis #to check there's no significant difference between rarefied and non-rarefied data qiime diversity beta-group-significance \ --i-distance-matrix bray_curtis_distance_matrix.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column status \ --o-visualization braycurtis_distance_res_status_rarefy.qzv \ --p-pairwise #5-6d alpha diversities qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table-72hrs.qza \ --p-sampling-depth 2359 \ --m-metadata-file sample-metadata.tsv \ --output-dir rarefied-core-metrics-results-72hrs qiime diversity alpha-group-significance \ --i-alpha-diversity rarefied-core-metrics-results-72hrs/shannon_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization rarefied-core-metrics-results-72hrs/shannon_vector.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity rarefied-core-metrics-results-72hrs/faith_pd_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization rarefied-core-metrics-results-72hrs/faith_pd_vector.qzv qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table-phyla-no-mt-no-infrequent.qza \ --p-sampling-depth 2359 \ --m-metadata-file sample-metadata.tsv \ --output-dir rarefied-core-metrics-results-all qiime diversity alpha-group-significance \ --i-alpha-diversity rarefied-core-metrics-results-all/shannon_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization rarefied-core-metrics-results-all/shannon_vector.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity rarefied-core-metrics-results-all/faith_pd_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization rarefied-core-metrics-results-all/faith_pd_vector.qzv #pairways permanova comparing deltamethrin exposed and unexposed (controls) #using both 2-3d and 5-6d mosquitoes qiime diversity beta \ --i-table table-phyla-no-mt-no-infrequent.qza \ --p-metric braycurtis \ --o-distance-matrix braycurtis_distance_matrix_all.qza qiime diversity beta-group-significance \ --i-distance-matrix braycurtis_distance_matrix_all.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column group \ --o-visualization braycurtis_distance_res_status_all.qzv \ --p-pairwise qiime diversity beta \ --i-table table-72hrs.qza \ --p-metric braycurtis \ --o-distance-matrix braycurtis_distance_matrix_72.qza qiime diversity beta-group-significance \ --i-distance-matrix braycurtis_distance_matrix_72.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column exposure \ --o-visualization braycurtis_distance_exposure_72.qzv \ --p-pairwise #pairways permanova looking at effect of age qiime diversity beta-group-significance \ --i-distance-matrix braycurtis_distance_matrix_all.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column age \ --o-visualization braycurtis_distance_res_age_all.qzv \ --p-pairwise #pairways permanova looking at effect of concentration in 2-3d qiime diversity beta-group-significance \ --i-distance-matrix braycurtis_distance_matrix.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column group \ --o-visualization braycurtis_distance_60_group.qzv \ --p-pairwise #Differential abundance ANCOM #2-3d only qiime composition add-pseudocount \ --i-table table-60mins.qza \ --o-composition-table comp-table-60mins.qza qiime composition ancom \ --i-table comp-table-60mins.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column status \ --o-visualization ancom-status.qzv #Differential abundance SONGBIRD #2-3 day mosquitoes #using Susceptible as the reference #i.e. resistant and control mosquitoes will be compared to susceptible qiime songbird multinomial \ --i-table table-60mins.qza \ --m-metadata-file sample-metadata.tsv \ --p-formula "C(status, Treatment('susceptible'))" --p-epochs 10000 \ --p-differential-prior 0.5 \ --p-summary-interval 1 \ --p-min-feature-count 10 \ --p-training-column train-or-test \ --p-num-random-test-examples 15 \ --o-differentials differentials_status_sus.qza \ --o-regression-stats regression-stats_status_sus.qza \ --o-regression-biplot regression-biplot_status_sus.qza #generate a null hypothesis to test the fit of the model qiime songbird multinomial \ --i-table table-60mins.qza \ --m-metadata-file sample-metadata.tsv \ --p-formula "1" \ --p-epochs 10000 \ --p-differential-prior 0.5 \ --p-summary-interval 1 \ --p-min-feature-count 10 \ --p-training-column train-or-test \ --p-num-random-test-examples 15 \ --o-differentials null-diff3.qza \ --o-regression-stats null-stats3.qza \ --o-regression-biplot null-biplot3.qza #this paired analysis compares the model with the null hypothesis qiime songbird summarize-paired \ --i-regression-stats regression-stats_sus.qza \ --i-baseline-stats null-stats3.qza \ --o-visualization paired-summary_status_null_sus.qzv #data is visualised in qurro qiime qurro differential-plot \ --i-table table-60mins.qza \ --i-ranks differentials_status_sus.qza \ --m-sample-metadata-file sample-metadata.tsv \ --m-feature-metadata-file feature-metadata.csv \ --o-visualization qurro-plot_status_sus.qzv