Here we show how to use WellReader with an example taken from experiments with fluorescent and luminescent reporters of the gene fis in E. coli.
After launching the application, download exp_2006_12_21b.CSV and open it in WellReader using the menus: File > Import > Raw data file (.csv).
The file contains three independent absorbance measurements, and thus you will be asked to merge these following the schema below. All measurements names should be identical or left empty.
Next, you are given the opportunity to enter some information about the experiment.
More info: see ImportExport
In this tutorial, we will work on the C1 well, containing fis expression data obtained with the fusion of the fis promoter to a luminescent reporter system. The backgrounds for the absorbance and luminescent measurements are contained in wells B10 and B11, respectively.
Using the mouse, define the following backgrounds:
Notice that the absorbance levels of both the target well C1 and the luminescence background well B11 are corrected by means of the data in B10.
When the backgrounds have been defined, press OK.
More info: see BackgroundDefinitionWindow, BackgroundCorrectionWindow
The final step before entering the main window is to detect erratic measurements called outliers.
You can select the measurements to which you want the automatic outlier detection function to be applied. Click the measurements in the list (use the Ctrl key to select multiple measurements) and press the Launch button. You can adjust the parameters for the automatic detection function but default values generally give good results.
As this step can take a few minutes on slow computers, it is possible to skip this step by pushing the Skip detection button.
For this tutorial select the abs1 measure and apply the automatic detection procedure with default parameters.
Upon completion of outlier detection, the MainWindow is shown.
The first step in analyzing fis expression data consists in analyzing the background absorbance. Select the absorbance background well B11.
In this particular case, the preview shows us that there are no outliers. However, we need to adjust the parameters for the spline fit. Click on the Fit data button.
As you can see, the smoothing parameter is far too high. Reduce it by a factor of 100 by clicking twice on the + button. Now things look better:
In the case of well B11, we are only interested in the absorbance measurement, so we ignore the other data for this well. Using the spline fit to the absorbance background data, we can now adjust the absorbance data in the other wells.
More info: see FitWindow
The next step again involves the analysis of a background. This time, we are concnerned with the luminescence background, defined as the luminescence level in well B10. If you select RLU in the preview window, you will see that we need to clean up outliers. Click on the Detect outliers button and a new window shows up. As we have applied the automatic detection for outliers when importing the data, some values have already been flagged as outliers.
You can modify the flagged values by drawing rectangles with the left mouse button or click on data points to select them. You can also deselect data points either by clicking a second time, or by drawing rectangles with the right mouse button. To precisely select or deselect data points you can use the zoom in and zoom out tools. To do this, either draw a rectangle with the SHIFT key hold, or right click and use the context menu. Once you have obtained the right level of details, select the outliers. In order to have a global view of the measurements, you can reset the zoom with the context menu. When you have finished the outlier detection, close the window by pressing the close button. In the preview window, you may choose to hide outliers and see the resulting data.
You can now adjust the spline fit, as for well B11. Do not forget to adjust both the absorbance and luminescence data! In fact, the absorbance measurements in the luminescence background well will be used for the background correction of the data for the gene fis. The RLU measurements do not need any adjustment, but the 'fit' parameter should be divided by 100.
Now that all necessary background measurements have been corrected, we can work on the well we are interested in, C1.
More info: see OutlierDetectionWindow, FitWindow, MainWindow#Wellpreview
For well C1, we again need to correct for absorbance and luminescence backgrounds by means of the data in wells B10 and B11. Detect outliers and adjust smoothing parameters as shown above. Then click on the Correct background button.
Absorbance measurements are available for both this well and the luminescence background well B10. This can be exploited for the refinement of background correction beyond simple subtraction (see BackgroundCorrection?). Changing the calibration parameters adjusts the growth rates of the cultures in the two wells, thus making the results better comparable as shown below.
This finishes the data preprocessing stage. We can now compute promoter activities and protein concentrations, which are the biologically meaningful quantities in the interpretation of the experimental data.
More info: see BackgroundCorrectionWindow
This last step is the easiest one. By clicking on the buttons labeled 'Compute promoter activity' and 'Compute concentration' on the bottom right part of the MainWindow opens new windows. The computed curves show the promoter activity as well as the reporter and the host protein concentrations computed from the data.
You may right click on each graph to export data if you want to play with it outside WellReader, for instance in MATLAB.
More info: see PromoterActivityWindow, ConcentrationWindow
