Data Exploration

Get Started

1) Register for a Galaxy account at https://usegalaxy.org/

2) Login

Note: Check if Galaxy is operational https://status.galaxyproject.org/

We will be using DESeq2 to perform differential gene expression analysis. More information about DESeq2 can be explored at the bottom of this page. EdgeR, limma, and trinity are other commonly used tools for DEG analysis. Read more about them (galaxy lists their publication references at the bottom webpage for each tool) in order to find the best tool for your future research!

Launch DESeq2

Use the search bar on the left side to find the software tool called 'DESeq2'.
- It is also under Genomics Analysis > RNA-seq > DESeq2
Click on the DESeq2 result to open the tool.
Tool opens in the middle pane.
See end of this page for further DESeq2 reading

Input count data

Factors correspond to sample comparisons we want to make. In this example, it is Arabidopsis meristem (Factor 1) vs leaf (Factor 2) samples.

1) Fill in the factor names where the boxes are.

2) Select the blue highlighted folder button to upload the data sets to the appropriate factors.

Upload Salmon Generated Count Data

After you click the folder button, a pop-up will appear. Click the 'Upload' button on the bottom left side.
Drag and drop your 3 Arabidopsis meristem quant files into this pop-up. Remember that the quantification files were the output files from Salmon.
Select '.txt' from the drop down menu for file type (highlighted in magenta). Note that the box is searchable so you don't have to scroll through the entire list.
Click the 'Start' button highlighted in blue. Once the files have uploaded, the rows will turn green and the Status will be 100%.
Click the 'Select' button. OR click outside of the pop up and the file will be in a right handed panel that you can now drag and drop into the counts files box for the appropriate factor level.
Repeat for the two Leaf samples in factor level two.

Set-up options

Select the DESeq2 options match these:

1) TPM values (magenta line)

2) Salmon for Program used (forest green line)

3) GTF/GFF3 for Gene mapping format (lime green line)

4) Then upload the TAIR10.E41.protein_coding.gtf for the Annotation file (blue line).

Note that this NEEDS to be the same gtf annotation file we used during Salmon quantification.

Submit the job!

Hit the execute button to submit your job!

Your job will appear in the right hand column.

Your job will look gray while it is waiting in the queue.
Your job will turn yellow when it starts running.
It will turn green once it is done!
Click the job to see the outputs and to download the files.

Performing separate analysis on Arabidopsis and Tomato

On the right hand menu, you can name you analysis as a "History." Rename your HISTORY as Arabidopsis DEG and then create a new history by clicking the plus sign '+' icon and you can then perfom DESeq2 using a new dataset for Tomato. Be sure to rename that HISTORY as well and now you can easy bounce back and forth between your separate experiments.

Supplemental Information About DESeq2

1) Concise R based tutorial of DESeq2 https://www.reneshbedre.com/blog/deseq2.html

2) More involved DESeq2 tutorial and information https://hbctraining.github.io/DGE_workshop/lessons/05_DGE_DESeq2_analysis2.html#:~:text=In%20DESeq2%2C%20the%20p%2Dvalues,used%20to%20determine%20significant%20genes.

3) Original publication of DESeq2 https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8

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