Importing example data for running Tertiary Analysis

If you haven’t run the Primary analysis and would rather download the example counts file to run the Tertiary analysis workflow, please follow the steps below -

https://zenodo.org/record/4541751/files/GSM461176_untreat_single.counts
https://zenodo.org/record/4541751/files/GSM461179_treat_single.counts
https://zenodo.org/record/4541751/files/GSM461182_untreat_single.counts

Note

  1. Import the three count files from Zenodo or the Shared Data library by pasting the above lines into the Upload data > “Paste/Fetch data” and clicking “Start”

  2. Rename each item so that it has the sample name, and the treatment - so for example - GSM461176_untreat_single.counts becomes GSM461176_untreat. To rename your sample, go to your sample name, and click on the pencil edit button next to the name and it will open the attributes of your sample. Go to the name, edit it and click “Save”

  3. Now, create a collection by clicking on the checkbox at the top of the history. Now, select all the 4 samples that you uploaded in the step 1. Under the settings button, select “Build Dataset List” and in the new textbox, enter a name for your collection and then click on “Create List” and then click on the checkbox at the top of your history again

  4. You will need three more files - “KEGG pathways” to provide pathways to goseq, “header” file to create a header file to attach to DESeq2, and the Drosophila gene annotation file to run this workflow.

  5. For the Drosophila gene annotation file, download the file “Drosophila_melanogaster.BDGP6.87.gtf” and upload it to the history

  6. For the header file, download the file (Above “History”, click on the three dots and download the file) from this page and upload the file to Galaxy using instructions from here

  7. For the KEGG pathway file, download the file from this page and upload the file to Galaxy using instructions from here

  8. You can now ready to run the Tertiary analysis workflow and go through the next pages to improve your understanding of each step

  9. Go to workflows > “RNA-seq Tertiary Analysis” and click on the run button

  10. Under “Input Dataset Collection”, select your collection of counts files

  11. Under GTF file - gene annotation, select your gene annotation file from your history

  12. Under “KEGG pathways to plot”, select the KEGG pathway file from your history

  13. Under “header”, select the header file from your history

  14. Another important note is for the “goseq” tool and “Pathview” tool. Depending on the gene ID format in your GTF file, scroll down to the two goseq tools in the workflow, expand each, and check the format under “Select Gene ID format”. Use the button next to it to select your gene ID format from the dropdown list. Repeat this for the “Pathview” tool as well

  15. Before you run the workflow, please review the changes you have to make to the DESeq2 tool instructions in order to run it with your dataset

  16. Scroll to the top and select “Yes” to send the results to a new history in order to send your workflow results to a new history and rename it so it’s intuitive to you

  17. Click on “Run Workflow”