- Click on the "Add" button
- A directory will open and you can search for the fastq.gz files you want to unzip
- Click on "OK" and it will load the files you chose
- The "Names" box will load all the files you inputed into the box
- I keep the settings as the default: If you want to change the setting, read the manual of GUNZIP here:Uncompress files with gunzip 1.6-2
- Click "Launch Analysis" once you are ready for the computer to start the analysis. (Repeat steps 1-4 as necessary until you have all the files you want loaded)
- In your analyses folder, click on "HTSeq-count-0.6.1"
- You will see "paired.sorted.XXXX.txt" files for your counts . This will be your count matrix
Example of Count Matrix for UT_top5M_1.sorted
Now that we know the quantity of each transcript in each sample we need to compare those quantities between samples. For this we will use DESeq2.
- Move all your paired.sorted.XXX.txt files to one folder for the easiest analysis
- Select all your paired.sorted.XXX.txt files for your input (you can not do 27 pairwise at the same time, select smaller sets of samples for your pairwise comparisons)
- Need to create a target file: Refer to https://pods.iplantcollaborative.org/wiki/pages/viewpage.action?pageId=28115144b look below for image
- Reference Biological condition: probably whatever the name is for your untreated/control samples
- Everything else: as default
Output File Types:
- In your analyses folder, click on "Deseq2(multifactorial pairwise comparison)"
- You will see "XXX.complete.txt" which has all the genes and your pairwise comparison
- You will see "XXX.up.txt" are genes that are upregulated in your pairwise comoparison
- You will see "XXX.down.txt" are genes that are downregulated in your pairwise comparison