Adding Annotation to Data

When you leave the Import run page, you are redirected to the Sample target list page, which gives you an overview of the targets (= genes) and samples qbase+ detected when reading in the datafiles. Take a look at the data. You see that the list of samples and targets matches the description of the qPCR experiment at the top of this page. The samples in this experiment are divided into two groups: samples that received some kind of treatment and untreated control samples. This information was not included in the run files so qbase+ does not know which sample belongs to which group. However, this is relevant information: in our analysis we are going to compare the expression of our genes of interest between treated and untreated samples. This means that qbase+ needs the grouping annotation to be able to perform the analysis we want to do. So we have to give qbase+ this annotation: we can do this by adding a custom sample property. To do this we need to create a sample properties file with a specific format that is described in the tutorial. You can find the file in the qbase+ folder on the BITS laptops or you can download the file here.

How to add the grouping annotation?

To import the file containing to grouping annotation:

• select Add samples and targets
• click Import sample list
• browse to the folder that contains the samples file
• select the file and click Open
• click Next

In the Importing samples window, you have to tell qbase+ which sample annotation you want to import from the sample properties file

In our case we could import Quantities (this annnotation is available in the sample properties file) but the quantities of the standard samples were included in the run files so qbase+ has already imported this annotation from the run files during data import. We definitely need to import the Custom properties since they were not a part of the run files. The Treatment property will tell qbase+ which samples belong to the group of control samples and which samples belong to the group of treated samples. Click the Next button at the bottom of the page to finish the import.

At this point you don’t see the custom annotation that you have imported, you will see it later in the analysis during scaling Leaving the Sample target list page takes you to the Run annotation page, where you have to confirm again that the sample and gene names are ok. If this is not the case you can adjust the annotation here.

Click the Next button at the bottom of the page

Our data file contains all required annotation:

• Cq values
• sample and target names
• sample types
• quantities for the standard samples
• grouping of the samples

Once runs are imported, you can start analyzing the data. Data consist of Cq values for all the wells.