Abel Thomas

Section 21

4/18/19

Purpose: The purpose of this lab was to continue with the qiime2 procedure and help us see the difference in the soil and the DNA found from both.  We were able to use more coding to help us see the exact difference in the barcodes of both soil types.

Purpose:

1. First we opened up terminal and made sure we still had qiime, if we did not then we redownloaded it
   1. Use “source activate qiime2-2019.1” to start qiime
2. Download the CILICURE_2018 folder from the Box which is linked in the powerpoint in the module section
3. Copy the folder from your Finder by double clicking
4. Change directory by using
   1. “cd /Users/abelthomas/Downloads/CILICURE_2018”
5. Add folders and sequences using the code
   1. qiime tools import \–type EMPPairedEndSequences \–input-path emp-paired-end-sequences \

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

6. Then we do demultiplexing which separates each barcode into its own
   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 \

      qiime demux summarize \

      –i-data demux.qza \

      –o-visualization demux.qzv

7. Then we denoised to 220 so we could make sure the DNA was small but still significant to see that there is a difference between the 2. Denoising cuts the sequence to the amount you said it to.
   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

      qiime feature-table summarize \

      –i-table table.qza \

      –o-visualization table.qzv \

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

      qiime feature-table tabulate-seqs \

      –i-data rep-seqs.qza \

      –o-visualization rep-seqs.qzv

      qiime metadata tabulate \

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

      –o-visualization denoising-stats.qzv

8. Then we created a phylogenetic tree to help show the relation between the organisms.
   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

9. After that step, we specified the phylogenetic tree by making files to organize the tree into the taxa and help show the concentrations of each organism in the soil.
   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

      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

Conclusion/Future Goals: After all these steps were completed then we were able to drag all the qzv files over onto Qiime2 View and see what the files showed us, the biggest one was the taxanomic bar plot which showed us the concentrations of ciliates found in each soil type. Then we were able to view all the sequences use the rep seqs qzv where we picked our sequence and ran it through BLAST and then found a PubMeds article on it. For the future we could use this for our later work with our own soil samples and find what we see from that.