Checking the Quality of Technical Replicates and Controls

The Technical quality control page handles the settings of the requirements that the data have to meet to be considered high quality. For instance the maximum difference between technical replicates is defined on this page. If there are technical replicates in the data set, qbase+ will detect them automatically (they have the same sample and target name) and calculate the average Cq value. In theory, technical replicates should generate more or less identical signals.

How to set the maximum difference in Cq values for technical replicates?

The quality criterium that the replicates must meet to be included for further analysis is one of the parameters in qbase+. You can set it on the Technical quality control page:

The default maximum allowed difference in Cq values between technical replicates is 0.5

Additionally, you can do quality checks based on the data of the positive and negative controls. |

How to set quality requirements for the control samples?

On the same Technical quality control page you can define the minimum requirements for a well to be included in the calculations:

• Negative control threshold : minimum allowed difference in Cq value between the sample with the highest Cq value and the negative control with the lowest Cq value: the default is 5 which means that negative controls should be more than 5 cycles away from the sample of interest.
• Lower and upper boundary : allowed range of Cq values for positive controls.

Excluded means that the data are ignored in the calculations.

How to check if there are wells that do not meet these criteria?

You can see flagged and excluded data by ticking the Show details… options on the Technical quality control page and clicking the Next button (purple) at the bottom of the page.

Qbase+ will open the results of the quality checks for the replicates and the controls on two different tabs. These tabs show lists of samples that failed the quality control criteria. When you open the replicates tab you can get an overview of the flagged or the excluded (purple) wells. Select the failing wells.

When the difference in Cq between technical replicates exceeds 0.5, the wells end up in the flagged or failing list. They are included in calculations unless you exclude them by unticking them. You see that the two replicates of Palm in Sample05 have very different Cq values. All other bad replicates are coming from standard samples. If you are finished checking the data quality, click Next to go to the Amplification efficiencies page.