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NGS Data RNA-seq T-BioInfo Interface
Grosmannia clavigera
Document Video

RNA-Seq Analysis with Grosmannia clavigera – Part 2, Process

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The first thing we will do as we go about the analysis is look at this section on top.

As a default, this section works with an annotated model genome. You will see several options of genomes that are pre-loaded on the platform. In our case, we will be using the RGenome function, meaning that the genome is not annotated. The genome file will have to be uploaded at the end of this process.

The data format we will use is fastQ and we will use Single End data.

In order to upload two datasets, correlating with 2 experiments that we will compare, we will have to use 2 groups and specify each group to contain 1 file.

Now we are ready to select our pipeline. The buttons that are highlighted are the possibilities after a module is selected. We can start by pressing on “data input”.

For now, we can ignore specific parameters for these modules and follow the preset values, click save after seeing this pop-up.

Let’s take a look at the TBioinfo interface again in detail

Every time a button is pressed, you will see an informational pop-up that also allows you to modify some parameters for the corresponding algorithm.

As you can see, once we completed data input, a number of options will become available. We will now select TopHat, a mapping algorithm.

Once we select TopHat, we will have one primary option available except for visualization. We will select CuffLinks since we are interested in differential expression. This algorithm builds isoforms for our data.

Once the isoforms are built, we need to integrate the data into a GTF file. CuffMerge.

Now, we have 2 options, one related to expression and the other to differential expression. We will select Cuffdiff that will compare expression data.

Once we are finished, we can click on “data output” and proceed to upload data.

To upload NGS data, you can either store the files on your computer and upload through this dialogue window, or copy the download links (such as from the SRA database) into a url.plst file with links. The other file that needs to be uploaded is the Genome file, under “Upload References”. The files can now be seen in the window.

Now the pipeline is ready to be run, so we can select the “Run Pipeline” button. This will be listed under “my pipelines”. Here the pipeline status with a percentage of the job done will be shown.