cd rmkdir /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit mkdir fastq #moved .fastq files into fastq directory, gzip them mkdir run1 # import manafest file from gdrive fetch-gdrive-object.py -d 1oA5EOIIRnTnpkSW6YtyclQBmipmt4doy7d7cik28PQ0 -t sheet -f tsv -o Skalanes-Test-Pit-Manafest.tsv conda activate qiime2-2020.2 source tab-qiime # in fastq folder qiime tools import \ --type 'SampleData[PairedEndSequencesWithQuality]' \ --input-path /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit/run1/Skalanes-Test-Pit-Manafest.tsv \ --output-path /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit/run1/Iceland-2019-STP-raw.qza \ --input-format PairedEndFastqManifestPhred33V2 # in run1 folder qiime demux summarize \ --i-data Iceland-2019-STP-raw.qza \ --o-visualization Iceland-2019-STP-raw.qzv cd /cluster/fieldscience/artifacts/Iceland-16S mkdir 2019-Skalanes-Test-Pit-run1 # use FWDs only fetch-gdrive-object.py -d 1Hnu-4gPQfmLv6Fb-n5E8Gpg7dcCnQz84F9uEHxF4XnQ -t sheet -f tsv -o Skalanes-Test-Pit-Manafest-FWDs.tsv # in fastq folder qiime tools import \ --type 'SampleData[SequencesWithQuality]' \ --input-path /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit/run1/Skalanes-Test-Pit-Manafest-FWDs.tsv \ --output-path /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit/run1/Iceland-2019-STP-FWDs-run1-raw.qza \ --input-format SingleEndFastqManifestPhred33V2 # in run1 folder qiime demux summarize \ --i-data Iceland-2019-STP-FWDs-run1-raw.qza \ --o-visualization Iceland-2019-STP-FWDs-run1-raw.qzv cp Iceland-2019-STP-FWDs-run1-raw.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 # trunc at 256 (quality score below 25) and trim at 31 to remove primer (did not check to see if primer was present) - leaves 225 bp nohup qiime dada2 denoise-single \ --p-n-threads 8 \ --i-demultiplexed-seqs Iceland-2019-STP-FWDs-run1-raw.qza \ --p-trunc-len 256 \ --p-trim-left 31 \ --o-representative-sequences Iceland-2019-STP-FWDs-rep-seqs-dada2-run1.qza \ --o-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --o-denoising-stats Iceland-2019-STP-FWDs-stats-dada2-run1.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-STP-FWDs-stats-dada2-run1.qza \ --o-visualization Iceland-2019-STP-FWDs-stats-dada2-run1.qzv # Move the .qzv file to the apache web browser: cp Iceland-2019-STP-FWDs-stats-dada2-run1.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 # get metadata file from drive fetch-gdrive-object.py -d 1wQ3XPlk0CN7bIoiAifaVTu-wNjsc91IdTTFByCKgkAY -t sheet -f tsv -o Skalanes-Test-Pit-Metadata.tsv # generate feature summaries qiime feature-table summarize \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --o-visualization Iceland-2019-STPs-FWDs-table-dada2-run1.qzv \ --m-sample-metadata-file Skalanes-Test-Pit-Metadata.tsv # Move the .qzv file to the apache web browser: cp Iceland-2019-STPs-FWDs-table-dada2-run1.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 # on average we kept 75% of reads after DADA2 without lowering the pmin - this is better than our Solo samples # minimum number of reads in lowest sample is 39066 - Retained 625,056 (71.99%) features in 16 (94.12%) samples at the specifed sampling depth. # average # of reads after DADA is 54264 # 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-STP-FWDs-rep-seqs-dada2-run1.qza \ --o-alignment Iceland-2019-STP-aligned-rep-seqs-dada2-run1.qza \ --o-masked-alignment Iceland-2019-STP-masked-aligned-rep-seqs-dada2-run1.qza \ --o-tree Iceland-2019-STP-unrooted-tree-run1.qza \ --o-rooted-tree Iceland-2019-STP-rooted-tree-run1.qza & qiime diversity core-metrics-phylogenetic \ --i-phylogeny Iceland-2019-STP-rooted-tree-run1.qza \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --p-sampling-depth 39066 \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --output-dir Iceland-2019-STP-core-metrics-run1 qiime diversity alpha-rarefaction \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --i-phylogeny Iceland-2019-STP-rooted-tree-run1.qza \ --p-max-depth 54264 \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-STP-alpha-rarefaction-run1.qzv cp Iceland-2019-STP-core-metrics-run1 /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 # Alpha Diversity metrics qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-STP-core-metrics-run1/faith_pd_vector.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-STP-core-metrics-run1/Iceland-2019-STP-faith-pd-group-significance-run1.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-STP-core-metrics-run1/evenness_vector.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-STP-core-metrics-run1/Iceland-2019-STP-evenness-group-significance-run1.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-STP-FWDs-rep-seqs-dada2-run1.qza \ --o-classification Iceland-2019-STP-taxonomy-silva-run1.qza & qiime metadata tabulate \ --m-input-file Iceland-2019-STP-taxonomy-silva-run1.qza \ --m-input-file Iceland-2019-STP-FWDs-rep-seqs-dada2-run1.qza \ --o-visualization Iceland-2019-STP-taxonomy-silva-table-run1.qzv qiime taxa barplot \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --i-taxonomy Iceland-2019-STP-taxonomy-silva-run1.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-rSTP-taxonomy-silva-bar-plots-run1.qzv #### Re-run with updated SILVA138 classifier and Qiime v2021.4 conda activate qiime2-2021.4 source tab-qiime cp silva-138-99-nb-classifier.qza /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit/run1 cd /mounts/pollock/16S/Iceland-2019/Skalanes-Test-Pit/run1 rm nohup.out nohup qiime feature-classifier classify-sklearn \ --p-n-jobs 8 \ --i-classifier silva-138-99-nb-classifier.qza \ --i-reads Iceland-2019-STP-FWDs-rep-seqs-dada2-run1.qza \ --o-classification Iceland-2019-STP-taxonomy-silva138-run1.qza & qiime metadata tabulate \ --m-input-file Iceland-2019-STP-taxonomy-silva-run1.qza \ --m-input-file Iceland-2019-STP-FWDs-rep-seqs-dada2-run1.qza \ --o-visualization Iceland-2019-STP-taxonomy-silva138-table-run1.qzv qiime taxa barplot \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --i-taxonomy Iceland-2019-STP-taxonomy-silva138-run1.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-rSTP-taxonomy-silva138-bar-plots-run1.qzv cp Iceland-2019-STP-taxonomy-silva138-table-run1.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 cp Iceland-2019-rSTP-taxonomy-silva138-bar-plots-run1.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 cp Iceland-2019-STP-taxonomy-silva138-run1.qza /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 ############################# # new and extra things from watching teh Qiime2 tutorial videos qiime diversity alpha-group-significance \ --i-alpha-diversity ./Iceland-2019-STP-core-metrics-run1/faith_pd_vector.qza \ --m-metadata-file ./Skalanes-Test-Pit-Metadata.tsv \ --o-visualization ./Iceland-2019-STP-core-metrics-run1/faiths_pd_statistics.qzv cp Iceland-2019-STP-core-metrics-run1/faiths_pd_statistics.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 qiime diversity alpha-group-significance \ --i-alpha-diversity ./Iceland-2019-STP-core-metrics-run1/evenness_vector.qza \ --m-metadata-file ./Skalanes-Test-Pit-Metadata.tsv \ --o-visualization ./Iceland-2019-STP-core-metrics-run1/evenness_statistics.qzv cp Iceland-2019-STP-core-metrics-run1/evenness_statistics.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 # rerun alpha diversity with metadata updated based on tephra layers - in a new folder fetch-gdrive-object.py -d 1wQ3XPlk0CN7bIoiAifaVTu-wNjsc91IdTTFByCKgkAY -t sheet -f tsv -o Skalanes-Test-Pit-Metadata.tsv mkdir run1-tephra qiime diversity core-metrics-phylogenetic \ --i-phylogeny Iceland-2019-STP-rooted-tree-run1.qza \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --p-sampling-depth 39066 \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --output-dir Iceland-2019-STP-core-metrics-run1-tephra qiime diversity alpha-rarefaction \ --i-table Iceland-2019-STP-FWDs-table-dada2-run1.qza \ --i-phylogeny Iceland-2019-STP-rooted-tree-run1.qza \ --p-max-depth 54264 \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-STP-alpha-rarefaction-run1-tephra.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-STP-core-metrics-run1-tephra/faith_pd_vector.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-STP-core-metrics-run1/Iceland-2019-STP-faith-pd-group-significance-run1-tephra.qzv qiime diversity alpha-group-significance \ --i-alpha-diversity Iceland-2019-STP-core-metrics-run1-tephra/evenness_vector.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --o-visualization Iceland-2019-STP-core-metrics-run1-tephra/Iceland-2019-STP-evenness-group-significance-run1-tephra.qzv # rename everything to have -tephra at the end cp Iceland-2019-STP-core-metrics-run1-tephra /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 qiime diversity alpha-group-significance \ --i-alpha-diversity ./Iceland-2019-STP-core-metrics-run1-tephra/faith_pd_vector-tephra.qza \ --m-metadata-file ./Skalanes-Test-Pit-Metadata.tsv \ --o-visualization ./Iceland-2019-STP-core-metrics-run1-tephra/faiths_pd_statistics-tephra.qzv cp Iceland-2019-STP-core-metrics-run1-tephra/faiths_pd_statistics-tephra.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 qiime diversity alpha-group-significance \ --i-alpha-diversity ./Iceland-2019-STP-core-metrics-run1-tephra/evenness_vector-tephra.qza \ --m-metadata-file ./Skalanes-Test-Pit-Metadata.tsv \ --o-visualization ./Iceland-2019-STP-core-metrics-run1-tephra/evenness_statistics-tephra.qzv cp Iceland-2019-STP-core-metrics-run1-tephra/evenness_statistics-tephra.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 # beta diversity qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-STP-core-metrics-run1-tephra/weighted_unifrac_distance_matrix-tephra.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --m-metadata-column area \ --o-visualization Iceland-2019-STP-core-metrics-run1-tephra/weighted_unifrac_distance_matrix-tephra.qzv \ cp Iceland-2019-STP-core-metrics-run1-tephra/weighted_unifrac_distance_matrix-tephra.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 qiime diversity beta-group-significance \ --i-distance-matrix Iceland-2019-STP-core-metrics-run1-tephra/bray_curtis_distance_matrix-tephra.qza \ --m-metadata-file Skalanes-Test-Pit-Metadata.tsv \ --m-metadata-column area \ --o-visualization Iceland-2019-STP-core-metrics-run1-tephra/bray_curtis_distance_matrix-tephra.qzv \ cp Iceland-2019-STP-core-metrics-run1-tephra/bray_curtis_distance_matrix-tephra.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1 ########################### getting raw fwd and rev for comparison with 2021 data for HardwareX paper ##################### # on Lovelace now cd /mounts/lovelace/16S/Iceland-2019/Skalanes-Test-Pit/run1 fetch-gdrive-object.py -d 1oA5EOIIRnTnpkSW6YtyclQBmipmt4doy7d7cik28PQ0 -t sheet -f tsv -o Iceland-2019-STP-manifest-FWD-REV.tsv qiime tools import \ --type 'SampleData[PairedEndSequencesWithQuality]' \ --input-path /mounts/lovelace/16S/Iceland-2019/Skalanes-Test-Pit/run1/Iceland-2019-STP-manifest-FWD-REV.tsv \ --output-path /mounts/lovelace/16S/Iceland-2019/Skalanes-Test-Pit/run1/Iceland-2019-STP-raw.qza \ --input-format PairedEndFastqManifestPhred33V2 qiime demux summarize \ --i-data Iceland-2019-STP-raw.qza \ --o-visualization Iceland-2019-STP-raw.qzv cp Iceland-2019-STP-raw.qzv /cluster/fieldscience/artifacts/Iceland-16S/2019-Skalanes-Test-Pit-run1