#========= Copying commands and scripts ========= mkdir -p $SCRATCH/NGS_metagenomics_Oct18/Scripts cd $SCRATCH/NGS_metagenomics_Oct18/Scripts cp /scratch/training/NGS_metagenomics/Oct18/*.sh . #========= Setting the environment ========= module load Anaconda/3-5.0.0.1 source activate qiime2-2018.8 module load R_tamu/3.4.2-intel-2017A-Python-2.7.12-default-mt #========= Importing Data ========= mkdir -p $SCRATCH/NGS_metagenomics_Oct18/Runs/FMT cd $SCRATCH/NGS_metagenomics_Oct18/Runs/FMT curl -sL "https://data.qiime2.org/2018.2/tutorials/fmt/sample_metadata.tsv" > "sample-metadata.tsv" curl -sL "https://data.qiime2.org/2018.2/tutorials/fmt/fmt-tutorial-demux-1-1p.qza" > "fmt-tutorial-demux-1.qza" curl -sL "https://data.qiime2.org/2018.2/tutorials/fmt/fmt-tutorial-demux-2-1p.qza" > "fmt-tutorial-demux-2.qza" #========= Sequence QC + Making Feature Table using DADA2 ========= #========= Summarization ========= qiime demux summarize \ --i-data fmt-tutorial-demux-1.qza \ --o-visualization demux-summary-1.qzv qiime demux summarize \ --i-data fmt-tutorial-demux-2.qza \ --o-visualization demux-summary-2.qzv #For visualizing any *.qzv file, you can download it locally and view it online at: https://view.qiime2.org #or run the command: "qiime tools view file.qzv" on HPRC portal > VNC session by logging into: portal.hprc.tamu.edu #========= DADA2 ========= #Sample QC using DADA2 R Package. Only SE, thus, ran for R1 and R2 files separately and then results are merged. qiime dada2 denoise-single \ --p-trim-left 13 \ --p-trunc-len 150 \ --i-demultiplexed-seqs fmt-tutorial-demux-1.qza \ --o-representative-sequences rep-seqs-1.qza \ --o-table table-1.qza \ --o-denoising-stats stats-1.qza qiime dada2 denoise-single \ --p-trim-left 13 \ --p-trunc-len 150 \ --i-demultiplexed-seqs fmt-tutorial-demux-2.qza \ --o-representative-sequences rep-seqs-2.qza \ --o-table table-2.qza \ --o-denoising-stats stats-2.qza #========= Merging ========= qiime feature-table merge \ --i-tables table-1.qza \ --i-tables table-2.qza \ --o-merged-table table.qza qiime feature-table summarize \ --i-table table.qza \ --o-visualization table.qzv \ --m-sample-metadata-file sample-metadata.tsv qiime feature-table merge-seqs \ --i-data rep-seqs-1.qza \ --i-data rep-seqs-2.qza \ --o-merged-data rep-seqs.qza qiime feature-table tabulate-seqs \ --i-data rep-seqs.qza \ --o-visualization rep-seqs.qzv #========= Creating tree for phylogenetic diversity analyses ========= qiime alignment mafft \ --i-sequences rep-seqs.qza \ --o-alignment aligned-rep-seqs.qza qiime alignment mask \ --i-alignment aligned-rep-seqs.qza \ --o-masked-alignment masked-aligned-rep-seqs.qza qiime phylogeny fasttree \ --i-alignment masked-aligned-rep-seqs.qza \ --o-tree unrooted-tree.qza qiime phylogeny midpoint-root \ --i-tree unrooted-tree.qza \ --o-rooted-tree rooted-tree.qza #========= Taxonomic analysis ========= curl -sL "https://data.qiime2.org/2018.2/common/gg-13-8-99-515-806-nb-classifier.qza" > "gg-13-8-99-515-806-nb-classifier.qza" qiime feature-classifier classify-sklearn \ --i-classifier gg-13-8-99-515-806-nb-classifier.qza \ --i-reads rep-seqs.qza \ --o-classification taxonomy.qza qiime metadata tabulate \ --m-input-file taxonomy.qza \ --o-visualization taxonomy.qzv qiime taxa barplot \ --i-table table.qza \ --i-taxonomy taxonomy.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization taxa-bar-plots.qzv #========= Alpha Diversity Analysis and Visualization ========= qiime diversity core-metrics-phylogenetic \ --i-phylogeny rooted-tree.qza \ --i-table table.qza \ --p-sampling-depth 84 \ --m-metadata-file sample-metadata.tsv \ --output-dir core-metrics-results qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization core-metrics-results/faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity core-metrics-results/evenness_vector.qza \ --m-metadata-file sample-metadata.tsv \ --o-visualization core-metrics-results/evenness-group-significance.qzv Testing command: qiime metadata tabulate \ --m-input-file core-metrics-results/faith_pd_vector.qza \ --o-visualization core-metrics-results/faith_pd_vector.qzv #========= Beta Diversity Analysis and Visualization ========= qiime diversity beta-group-significance \ --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column treatment-group \ --o-visualization core-metrics-results/unweighted-unifrac-treatment-group-significance.qzv \ --p-pairwise qiime diversity beta-group-significance \ --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file sample-metadata.tsv \ --m-metadata-column administration-route \ --o-visualization core-metrics-results/unweighted-unifrac-administration-route-significance.qzv \ --p-pairwise #========= Alpha rarefaction plotting ========= qiime diversity alpha-rarefaction \ --i-table table.qza \ --i-phylogeny rooted-tree.qza \ --p-max-depth 380 \ --m-metadata-file sample-metadata.tsv \ --o-visualization alpha-rarefaction.qzv