Log explaining processing of data and Prep for Qiime 2019.1 ################DISCLAIMER: commands are specific to QIIME2 version####################### ################ if you are working with another i.e. the latest version################## ################please refer to the help pages at qiime2.org############################## the following code moreover assumes that you are ok with files being written directly in the directory you are working in. If not, create new directories and make sure to specify the path you want output files to be written into. we recommend to make directories for "Artifacts", "Visualisations" and Stats outputs and writing the respective files into the directories. 1. import data qiime tools import \ --type 'SampleData[SequencesWithQuality]' \ --input-path path/to/Manifest \ --output-path Bloomi_paired-end-demux.qza \ --input-format PairedEndFastqManifestPhred33ΒΆ qiime demux summarize \ --i-data Bloomi_paired-end-demux.qza \ --o-visualization Bloomi_paired-end-demux.qzv 2. Denoiding with Dada2 since for our specific samples quality drops beyond 25 at position 242, this is where we will be cutting the reverse reads, we will also be removing the remaining primer sequences qiime dada2 denoise-paired \ --i-demultiplexed-seqs Bloomi_demux.qza \ --p-trunc-len-f 0 \ --p-trunc-len-r 242 \ --p-trim-left-f 15 \ --p-trim-left-r 20 \ --p-chimera-method consensus\ --o-representative-sequences 1_rep_seqs_bloomi.qza \ --o-table 1_table_bloomi.qza \ --o-denoising-stats 1_denoising_stats_bloomi.qza \ --verbose 3. generate a tree for phylogenetic diversity analyses qiime phylogeny align-to-tree-mafft-fasttree \ --i-sequences 1_rep_seqs_bloomi.qza \ --o-alignment 1.2_aligned_repseqs_bloomi.qza \ --o-masked-alignment 1.3.dada2_masked_alignment_bloomi.qza \ --o-tree 1.4.bloomi_unrooted_tree.qza \ --o-rooted-tree 1.5.bloomi_rooted_tree.qza 4. summarizing denoising stats to be able to view them metadata tabulate \ --m-input-file 1_denoising_stats_bloomi.qza \ --o-visualization 1_denoising_stats_bloomi.qzv 5. summarising feature table data for the feature table the metadata file corresponding to the samples needs to be prepared. the metadata file can be validated using Keemei (https://keemei.qiime2.org/) qiime feature-table summarize \ --i-table Artifacts/1_table_bloomi.qza \ --m-sample-metadata-file Bloomi_redMetadata.tsv \ --o-visualization Visualizations/1_bloomi_table.qzv 6. Taxonomic classification and training the classifier for convenience the necessary Artifacts for taxonomic classification have been added to the source files. For your own data, classifiers need to be trained on the used primers and we suggest using the latest taxonomy versions available (i.e. Silva or Greengenes) 6.1 fitting the classifier qiime feature-classifier fit-classifier-naive-bayes \ --i-reference-reads SILVA_99_ref_seqs.qza \ --i-reference-taxonomy SILVA_99otu_taxonomy.qza \ --o-classifier silva_bloomi_classifier.qza \ --verbose 6.2. applying the classifier to extract taxonomy of reference reads qiime feature-classifier classify-sklearn \ --i-classifier silva_bloomi_classifier.qza \ --i-reads 1_rep_seqs_bloomi.qza \ --o-classification bloomi_trained_taxonomy_silva_132_99_classified.qza \ --p-n-jobs -4 \ --verbose 6.3.tabulating the taxonomy qiime metadata tabulate \ --m-input-file bloomi_trained_taxonomy_silva_132_99_classified.qza \ --o-visualization bloomi_trained_taxonomy_silva_132_99_classified.qzv 7 Exporting Artifacts in order to be able to use them in Phyloseq and R for this a directory called export was created , the exported items need to be renamed 7.1. the feature table qiime tools export \ --input-path Artifacts/1_table_bloomi.qza \ --output-path export/ 7.2. export the taxonomy qiime tools export \ --input-path bloomi_trained_taxonomy_silva_132_99_classified.qza --output-path export/ 7.3. convert biom to tsv to txt biom convert -i export/feature-table.biom \ --o export/1_feature_bloomi.txt \ --to-tsv now change feature ID to OTUID in taxonomy.tsv using nano 7.4. export the unrooted tree qiime tools export \ --input-path Artifacts/1.4.1.4.bloomi_unrooted_tree.qza \ --output-path export/