# Create a new working directory in /mounts/pollock/16S/Iceland-2019 mkdir qiime2-run4-FWD-all-spots # Move the files from /cluster/metagenomes/Iceland-2019/Solo/16S/Casava-1.8a into the new working directory cp /cluster/metagenomes/Iceland-2019/Solo/16S/Casava-1.8a/spot-6_S6_L001_R1_001.fastq qiime2-run4-FWD-all-spots/ cp /cluster/metagenomes/Iceland-2019/Solo/16S/Casava-1.8a/spot-9_S9_L001_R1_001.fastq qiime2-run4-FWD-all-spots/ # Convert all .fastq into .fastq.gz gzip *.fastq # gzip all the files named *.fastq # Copy Silva classifier, Manifest and Metadata Files from pervious runs cp qiime2-run1/silva-132-99-nb-classifier.qza qiime2-run4-FWD-all-spots/ cp qiime2-run1/Iceland-2019-Solo-Metadata.tsv qiime2-run4-FWD-all-spots/ cp qiime2-run3-FWD-only/Iceland-2019-Solo-FWD-manifest.tsv qiime2-run4-FWD-all-spots/ # Log into Qiime2.0 on pollock conda activate qiime2-2020.2 source tab-qiime # Import the sequencing files into a .qza artifact qiime tools import \ --type 'SampleData[SequencesWithQuality]' \ --input-path Iceland-2019-Solo-FWD-manifest.tsv \ --output-path Iceland-2019-run4-single-end-demux.qza \ --input-format SingleEndFastqManifestPhred33V2 # Produce the summary .qzv of the raw data: qiime demux summarize \ --i-data Iceland-2019-run4-single-end-demux.qza \ --o-visualization Iceland-2019-run4-raw.qzv # Put the .qzv file into the apache web browser cd /cluster/fieldscience/artifacts mkdir Iceland-2019-run4-FWD-all-spots cd /mounts/pollock/16S/Iceland-2019/qiime2-run4-FWD-all-spots/ cp Iceland-2019-run4-raw.qzv /cluster/fieldscience/artifacts/Iceland-2019-run4-FWD-all-spots/ # Run DADA2 truncating at 298 for the FWD reads nohup qiime dada2 denoise-single \ --p-n-threads 8 \ --i-demultiplexed-seqs Iceland-2019-run4-single-end-demux.qza \ --p-trunc-len 298 \ --p-trim-left 0 \ --o-representative-sequences Iceland-2019-run4-rep-seqs-dada2.qza \ --o-table Iceland-2019-run4-table-dada2.qza \ --o-denoising-stats Iceland-2019-run4-stats-dada2.qza & # To check on the job, you can use ps to see how it’s running. # summarize resulting statistics qiime metadata tabulate \ --m-input-file Iceland-2019-run4-stats-dada2.qza \ --o-visualization Iceland-2019-run4-stats-dada2.qzv # Move the .qzv file to the apache web browser: cp Iceland-2019-run4-stats-dada2.qzv /cluster/fieldscience/artifacts/Iceland-2019-run4-FWD-all-spots # generate feature summaries qiime feature-table summarize \ --i-table Iceland-2019-run4-table-dada2.qza \ --o-visualization Iceland-2019-run4-table-dada2.qzv \ --m-sample-metadata-file Iceland-2019-Solo-Metadata.tsv # Move the .qzv file to the apache web browser: cp Iceland-2019-run4-table-dada2.qzv /cluster/fieldscience/artifacts/Iceland-2019-run4-FWD-all-spots # Create a phylogenetic tree from the data: rm nohup.out nohup qiime phylogeny align-to-tree-mafft-fasttree \ --p-n-threads 8 \ --i-sequences Iceland-2019-run4-rep-seqs-dada2.qza \ --o-alignment Iceland-2019-run4-aligned-rep-seqs-dada2.qza \ --o-masked-alignment Iceland-2019-run4-masked-aligned-rep-seqs-dada2.qza \ --o-tree Iceland-2019-run4-unrooted-tree.qza \ --o-rooted-tree Iceland-2019-run4-rooted-tree.qza & # Make sure to match --p-sampling-depth and --p-max-depth to your current data set # Retained 577,995 (44.30%) features in 11 (91.67%) samples at the specifed sampling depth (52545) qiime diversity core-metrics-phylogenetic \ --i-phylogeny Iceland-2019-run4-rooted-tree.qza \ --i-table Iceland-2019-run4-table-dada2.qza \ --p-sampling-depth 52545 \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --output-dir Iceland-2019-run4-core-metrics qiime diversity alpha-rarefaction \ --i-table Iceland-2019-run4-table-dada2.qza \ --i-phylogeny Iceland-2019-run4-rooted-tree.qza \ --p-max-depth 131464 \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-alpha-rarefaction.qzv # Alpha Diversity metrics qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-run4-core-metrics/faith_pd_vector.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-core-metrics/Iceland-2019-run4-faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-run4-core-metrics/evenness_vector.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-core-metrics/Iceland-2019-run4-evenness-group-significance.qzv # Beta Analysis qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-core-metrics/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --o-visualization Iceland-2019-run4-core-metrics/unweighted-unifrac-sector-significance.qzv \ qiime emperor plot \ --i-pcoa Iceland-2019-run4-core-metrics/unweighted_unifrac_pcoa_results.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-custom-axes year-of-last-ice \ --o-visualization Iceland-2019-run4-core-metrics/unweighted-unifrac-emperor-sector.qzv qiime emperor plot \ --i-pcoa Iceland-2019-run4-core-metrics/bray_curtis_pcoa_results.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-custom-axes year-of-last-ice \ --o-visualization Iceland-2019-run4-core-metrics/bray-curtis-emperor-sector.qzv # Taxonomic analysis rm nohup.out nohup qiime feature-classifier classify-sklearn \ --p-n-jobs 8 \ --i-classifier silva-132-99-nb-classifier.qza \ --i-reads Iceland-2019-run4-rep-seqs-dada2.qza \ --o-classification Iceland-2019-run4-taxonomy-silva.qza & qiime metadata tabulate \ --m-input-file Iceland-2019-run4-taxonomy-silva.qza \ --m-input-file Iceland-2019-run4-aligned-rep-seqs-dada2.qza \ --o-visualization Iceland-2019-run4-taxonomy-silva-table.qzv qiime taxa barplot \ --i-table Iceland-2019-run4-table-dada2.qza \ --i-taxonomy Iceland-2019-run4-taxonomy-silva.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-taxonomy-silva-bar-plots.qzv # Move everything onto the apache web browser cp *.qzv /cluster/fieldscience/artifacts/Iceland-2019-run4-FWD-all-spots # Filtering to remove mocks, spot 12 from the dataset mkdir filtered-data qiime feature-table filter-samples \ --i-table Iceland-2019-run4-table-dada2.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-where "[group] IN ('one', 'two', 'three')" \ --o-filtered-table filtered-data/Iceland-2019-run4-filtered-table-dada2.qza qiime feature-table summarize \ --i-table filtered-data/Iceland-2019-run4-filtered-table-dada2.qza \ --m-sample-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization filtered-data/Iceland-2019-run4-filtered-table-dada2.qzv cp filtered-data/Iceland-2019-run4-filtered-table-dada2.qzv /cluster/fieldscience/artifacts/Iceland-2019-run4-FWD-all-spots qiime diversity core-metrics-phylogenetic \ --i-phylogeny Iceland-2019-run4-rooted-tree.qza \ --i-table filtered-data/Iceland-2019-run4-filtered-table-dada2.qza \ --p-sampling-depth 52545 \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --output-dir Iceland-2019-run4-filtered-core-metrics qiime diversity alpha-rarefaction \ --i-table filtered-data/Iceland-2019-run4-filtered-table-dada2.qza \ --i-phylogeny Iceland-2019-run4-rooted-tree.qza \ --p-max-depth 119249 \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization filtered-data/Iceland-2019-run4-alpha-rarefaction.qzv # Filtered Alpha Diversity metrics qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-run4-filtered-core-metrics/faith_pd_vector.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-filtered-core-metrics/Iceland-2019-run4-filtered-faith-pd-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-run4-core-metrics/evenness_vector.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-filtered-core-metrics/Iceland-2019-run4-filtered-evenness-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-run4-core-metrics/shannon_vector.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-filtered-core-metrics/Iceland-2019-run4-filtered-shannon-group-significance.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-run4-core-metrics/observed_otus_vector.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization Iceland-2019-run4-filtered-core-metrics/Iceland-2019-run4-filtered-observed-otus-group-significance.qzv # Filtered Beta Diversity qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-filtered-core-metrics/weighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-pairwise \ --o-visualization Iceland-2019-run4-filtered-core-metrics/weighted-unifrac-sector-significance.qzv \ qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-filtered-core-metrics/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-pairwise \ --o-visualization Iceland-2019-run4-filtered-core-metrics/unweighted-unifrac-sector-significance.qzv \ qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-filtered-core-metrics/jaccard_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-pairwise \ --o-visualization Iceland-2019-run4-filtered-core-metrics/jaccard-sector-significance.qzv \ qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-filtered-core-metrics/bray_curtis_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-pairwise \ --o-visualization Iceland-2019-run4-filtered-core-metrics/bray-curtis-sector-significance.qzv \ qiime emperor plot \ --i-pcoa Iceland-2019-run4-filtered-core-metrics/bray_curtis_pcoa_results.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-custom-axes year-of-last-ice \ --o-visualization Iceland-2019-run4-filtered-core-metrics/bray-curtis-emperor-sector.qzv qiime emperor plot \ --i-pcoa Iceland-2019-run4-filtered-core-metrics/unweighted_unifrac_pcoa_results.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-custom-axes year-of-last-ice \ --o-visualization Iceland-2019-run4-filtered-core-metrics/unweighted-unifrac-emperor-sector.qzv qiime emperor plot \ --i-pcoa Iceland-2019-run4-filtered-core-metrics/weighted_unifrac_pcoa_results.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-custom-axes year-of-last-ice \ --o-visualization Iceland-2019-run4-filtered-core-metrics/weighted-unifrac-emperor-sector.qzv qiime emperor plot \ --i-pcoa Iceland-2019-run4-filtered-core-metrics/jaccard_pcoa_results.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-custom-axes year-of-last-ice \ --o-visualization Iceland-2019-run4-filtered-core-metrics/jaccard-emperor-sector.qzv # Filtered Taxonomic analysis qiime taxa barplot \ --i-table filtered-data/Iceland-2019-run4-filtered-table-dada2.qza \ --i-taxonomy Iceland-2019-run4-taxonomy-silva.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --o-visualization filtered-data/Iceland-2019-run4-filtered-taxonomy-silva-bar-plots.qzv # Move everything onto the apache web browser cp *.qzv /cluster/fieldscience/artifacts/Iceland-2019-run4-FWD-all-spots # Filtering tests # Test 1 - filter by different distance matricies - note this code didn't actually created filtered data ... not going to bother troubleshooting atm mkdir filtering-tests qiime diversity filter-distance-matrix \ --i-distance-matrix Iceland-2019-run4-core-metrics/jaccard_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-where "[group] IN ('one', 'two', 'three')" \ --o-filtered-distance-matrix filtering-tests/jaccard-filtered-distance-matrix.qza qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-core-metrics/jaccard_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-method 'permanova' \ --o-visualization filtering-tests/jaccard-sector-significance.qzv \ qiime diversity filter-distance-matrix \ --i-distance-matrix Iceland-2019-run4-core-metrics/bray_curtis_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-where "[group] IN ('one', 'two', 'three')" \ --o-filtered-distance-matrix filtering-tests/bray-curtis-filtered-distance-matrix.qza qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-core-metrics/bray_curtis_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-method 'permanova' \ --o-visualization filtering-tests/bray-curtis-sector-significance.qzv \ qiime diversity filter-distance-matrix \ --i-distance-matrix Iceland-2019-run4-core-metrics/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-where "[group] IN ('one', 'two', 'three')" \ --o-filtered-distance-matrix filtering-tests/unweighted-unifrac-filtered-distance-matrix.qza qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-core-metrics/unweighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-method 'permanova' \ --o-visualization filtering-tests/unweighted-unifrac-sector-significance.qzv \ qiime diversity filter-distance-matrix \ --i-distance-matrix Iceland-2019-run4-core-metrics/weighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --p-where "[group] IN ('one', 'two', 'three')" \ --o-filtered-distance-matrix filtering-tests/weighted-unifrac-filtered-distance-matrix.qza qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-run4-core-metrics/weighted_unifrac_distance_matrix.qza \ --m-metadata-file Iceland-2019-Solo-Metadata.tsv \ --m-metadata-column year-of-last-ice \ --p-method 'permanova' \ --o-visualization filtering-tests/weighted-unifrac-sector-significance.qzv \