Objective

• Continue using and getting comfortable with Qiime2
• Analyze our eDNA from the soil samples, both the Chelex and PowerSoil samples, through Qiime tools

Procedure

1. Retrieve the folder containing all the data from the Box link
2. Open Terminal and make sure Qiime2 is installed and running correctly
3. Activate Qiime2
   1. source activate qiime2-2019.1
4. Change the directory to the downloaded folder in the Terminal
   1. cd (copy and paste the folder into the Terminal)
5. Import sequences as Qiime2 artifact
   1. qiime tools import \

      –type EMPPairedEndSequences \

      –input-path emp-paired-end-sequences \

      –output-path emp-paired-end-sequences.qza

6. Demultiplex the sequence
   1. qiime demux emp-paired \

      –m-barcodes-file sample-metadata.tsv \

      –m-barcodes-column BarcodeSequence \

      –i-seqs emp-paired-end-sequences.qza \

      –o-per-sample-sequences demux.qza \

7. Create a summary for the demultiplexed sequences
   1. qiime demux summarize \

      –i-data demux.qza \

      –o-visualization demux.qzv

8. Denoise up to 220 bases
   1. qiime dada2 denoise-paired \

      –i-demultiplexed-seqs demux.qza \

      –p-trunc-len-f 220 \

      –p-trunc-len-r 220 \

      –o-table table.qza \

      –o-representative-sequences rep-seqs.qza \

      –o-denoising-stats denoising-stats.qza

9. Create a feature table to summarize and list all the sequences
   1. qiime feature-table summarize \

      –i-table table.qza \

      –o-visualization table.qzv \

      –m-sample-metadata-file sample-metadata.tsv

10. Tabulate the feature table
    1. qiime feature-table tabulate-seqs \

       –i-data rep-seqs.qza \

       –o-visualization rep-seqs.qzv

11. Visualize the stats of the denoising outcome
    1. qiime metadata tabulate \

       –m-input-file denoising-stats.qza \

       –o-visualization denoising-stats.qzv

12. Create a phylogenetic tree
    1. qiime phylogeny align-to-tree-mafft-fasttree \

       –i-sequences rep-seqs.qza \

       –o-alignment aligned-rep-seqs.qza \

       –o-masked-alignment masked-aligned-rep-seqs.qza \

       –o-tree unrooted-tree.qza \

       –o-rooted-tree rooted-tree.qza

13. Run the data against a reference database
    1. qiime feature-classifier classify-sklearn –i-classifier silva-132-99-515-806-nb-classifier.qza –i-reads rep-seqs.qza –o-classification taxonomy.qza
14. Tabulate a table with all the sequences and classification information
    1. qiime metadata tabulate \

       –m-input-file taxonomy.qza \

       –o-visualization taxonomy.qzv

15. Tabulate a barplot with all the classification information
    1. qiime taxa barplot \

       –i-table table.qza \

       –i-taxonomy taxonomy.qza \

       –m-metadata-file sample-metadata.tsv \

       –o-visualization taxa-bar-plots.qzv

Results

Demultiplexed:

Denoised:

Taxonomy Table:

Taxonomy Barplot:

Conclusion

I feel much more confident in working with Qiime2 and the Terminal. Running the commands is much more easier and I am beginning to understand what the different commands do and how the data is being processed.