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Galaxy DNA-Seq Tutorial

Linking to data

Link in the Mark Pritchard Vaccinia virus data set.

Start with a blank history, there should be no numbered items on the right hand side of the pane. Otherwise create a new history.
Select "Shared Data" from the top of the screen to bring up the Shared Data screen
Select Data Library
Select "Pritchard Vaccinia WR FastQ Files" from the alphabetically sorted list

Click on the subfolder top box to select all 6 files
Select "import to current history"
Click on "Analyze Data" from the upper main menu. It should bring up the main page and your history pane should now look like the image below.

Notice that with just 3 viruses are already over 4 GB of data!

Formatting and Grooming Data

Your data is often NOT what you expect

FastQ Format

FastQ format can mean many things, see the wikipedia entry on FastQ format
Ilumina FastQ format also not informative, it changes
Click on the pencil to icon in one of the virus images to pull up the attributes, your screen should look a bit like this:

In Galaxy the expected data type of the galaxy tool must match EXACTLY with the data type in your history pane, otherwise the option to use that particular piece of data will not appear in the tool's drop down menu for data selection.
Galaxy requires that everything go into Sanger format to be used. If you know your data is in sanger format, select fastqsanger for your data type. If it is not in that format, select fastq and run the FastQ Groomer.
FastQ groomer is very useful, but slow
Not a bad idea to run it if you are dealing with a new machine and have the time. Costly in terms of space.

Reference Genome

Ensure the correct reference genome is selected
WARNING - seeing the reference genome doesn't mean it is there for using
The current list of genomes available at UAB is here
Used for a variety of tools, it can be overridden earlier
Any problems with CMV-21950R_3_1.fastq ?

Assessing the quality of the data

We will use a number of different tools from the "NGS: QC and manipulation" drop down menu. Try processing the FastQ files with:

FastQC
Compute Quality Statistics

FastQC

FastQC gives an attractive visual output and will flag potential problems.

Select NGS: QC and manipulation -> FastQC

Run it on all the fastq files
Remember, you are on a cluster, this can be done in parrallel

Take a look at the other areas that show up as quality issues.

FastQC flags potential problems
Are there any problems for us?

It is imperative to understand your organism and its biology to interpret the data

Quality Statistics and Boxplot

Galaxy has its own set of tools for computing quality statistics (using R)
Generates raw statistics and a pretty box plot
Select NGS: QC and manipulation -> Compute Quality Statistics for CMV-21950R_3_1.fastq
Run it