Sara Rothrock

4/18/2019

Purpose:

The purpose of this lab was to analyze the soil eDNA we collected from our soil samples and extracted DNA from.

Procedure:

1. Before beginning eDNA analysis, ensure that Qiime2 is installed on the computer you will be using. If not downloaded, then download Qiime2 again.
2. After ensuring Qiime2 is downloaded, download the folder that contains all the data from the box link.
3. Activate Qiime2:
   • source activate Qiime2-2019.1
4. Switch the directory to the folder CILI-CURE_2018:
   • cd CILI-CURE_2018
5. Import sequences as Qiime2 artifact:
   • qiime tools import \–type EMPPairedEndSequences \

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

     –output-path emp-paired-end-sequences.qza
6. Demultiplex the sequences:
   • 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. Denoise using DADA2 to 220 bases:
   • 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

8. Create a Feature Table:
   • 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
9. Create a phylogenic tree:
   • 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

10. Create a chart with Taxonomic classification:
    • 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

Results:

Demultiplexed-

Denoised-

Taxonomy Table-

Taxonomy Barplot-

Conclusion:

Overall, this lab was very successful because the only issues I had was having to re-download the Qiime2 program onto the computer. I feel much more confident using Qiime2 and using computer terminals to download files. Also, I tried the bonus to try and understand the programming better and learn about new codes for alpha and beta. In this lab, I also discovered that there was a ciliate that was recently discovered in Northern China that was in our soil sample. The ciliate did not have a food vacuole and it’s diet seemed to be of smaller microorganisms. I thought this was extremely fascinating and I am excited to further analyze our data and see what other types of ciliates there are in our soil sample.

Future Steps:

In the future, I would like to continue learning how to do metabarcoding and to expand my knowledge on data analysis.