The PCA Window 

Select the node you wish to run the analysis on in the Project Tree, and then click the Principal Component Analysis (    ) button on the J-Express Pro tool bar. Alternatively, select Methods | Principal Component Analysis from the J-Express Pro menu bar.

The PCA window opens, and it follows the common pattern of most windows in J-Express Pro, with a menu and a tool bar, with an area below it for data display organized into tabs. When a PCA window is first opened it contains two tabs: PCA and T humbs.

The PCA tab

The PCA tab shows a 2D plot of the dataset. The axis chosen by default are the ones that result in the highest total variance. Each profile is represented in the PCA plot as a dot. Additionally, the density of dots in each local area is indicated by a range of colors (by default white (lowest density) through blue and red to yellow (highest density)). Thus high numbers of dots in an area will be obvious, even though the dots more or less overlap. If a dataset is large, or the data is centered in a relative small area, it is possible to define a threshold value. If the dot density exceeds this value, the dots will be removed in this area. This makes it easier to see the underlying structure of the spread of the plot, and identify and select outliers. The variance of the axis, and the total variance for the plot are displayed in the bottom left corner.  The color range for relative dot density is shown in the bottom right corner of the plot.

To focus on an area of interest in the PCA plot, click the Frame content to PCA(    ) and make sure that the Frame method is set to    Square. Drag out a selection box around the area by clicking and dragging with the mouse. The PCA plot will be zoomed to the selected area. Alternately you can select the Frame contents to chart button (    ). If the area contains any profiles, they will be added as a thumbnail to the Thumbs tab. Alternatively use the Lasso tool (    ) to draw the selection area. The lasso tool is found by clicking the Frame Method button and then selecting    Lasso. Different types of fill can also be chosen for the selected area. Further PCA operation will only affect the selected area.

Customizing the PCA plot

The PCA properties window.

To customize a PCA plot, select PCA | PCA Properties from the PCA menu bar. Another way to bring up the PCA properties window is right clicking on the PCA plot

Fill lets you choose the background color of the PCA plot. The options are:

• One color - click the Background color box to select a new background color for the PCA plot.

• Density map - uses a spectrum of colors to show the density of PCA points in an area.

Density Map options:

These options become available when the density map is selected as the fill type .

Density Map Colors - allows you to change the color of the highlights. To change a color in the PCA color range simply click one of the small boxes over the spectrum. This brings up a color selection dialog where you can choose the color you want. Click OK, and the color range will change to accommodate your changes.

Density area - allows you to set the size of the area a single dot influences on the density map. To make the influence of a dot less, move the slider to the left, to increase the influence of a dot move the slider to the right.

Number of Colors - sets the number of colors to be used to generate the density map.  A smaller number of colors limits, and in some cases removes the density map for dots lying in areas of low density. In addition the transition between colors becomes less gradual. Move the slider to set the desired amount of colors to be used.

Paint Threshold - sets a threshold value for the amount of dots in an area. If this threshold is exceeded the dots in that area are removed. This frequently helps show the structure of the Density Map. Move the slider to set the desired threshold.

• Gradient - Two colors are combined to create a smooth color gradient. Click the two colored boxes to choose the desired colors. Use the Gradient Type menu to select the type of gradient. Diagonal forms a color gradient from the upper left to the lower right corner; Top-Bottom forms a color gradient from the top of the plot to the bottom.

• External Picture - Use the file selection dialog to select the image file you wish to use as a background for the plot. Selecting Stretch will stretch the image to fit the plot. Selecting Tile will repeat the image in a tile pattern if it is too small to cover the entire plot.

• Tiles - Six additional patterns you can use for your plots.

Density Map

• Density Area - the value in the Density Area text field says how far out (in pixels) from a dot the density circle should stretch.

• Number of colors - the number of colors that will be used to draw the density areas.

• Paint threshold (%) - if certain areas are very dense, you may want to remove some of  these profiles from the plot. This makes it easier to for instance spot differentially expressed genes. If the threshold value is set to i.e. 10, only the profiles belonging to the 10% least dense areas will be plotted.

• Colors - click on the colored squares to change the colors.

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• Spot size lets you set the size in pixels of the PCA points.

• Circular Spots - check this box to use circular PCA points.

• Framed - Checking this box adds a frame around each dot.

• Title - enter a title for your chart in this box, if needed. It will appear at the top of the chart.

Axis Value Span lets you set the maximum and minimum values for each axis. Uncheck the Force Endlabels box to turn off the automatic endlabels generated by J-Express Pro. Click the Reset button to reset the value span.

Chart & Axis color - click these colored boxes to set the background color for the area outside the main chart, and the colors used for the axis.

X- and Y-axis options

• Title allows you to name each axis. The name will appear on the left side of the chart for the y-axis, and on the bottom of the chart for the x-axis.

• Minor tics sets the amount of minor tics between each major tic on the respective axis.

• Tics on both ends - check this box to have tics on the opposite edge of the plot from the axis, in addition to the tics on the axis.

Grid lets you set options for the plot grid.

• Paint Grid - check this box to toggle display of the grid on. Uncheck it to toggle display of the grid off.

• Grid Color - select the desired color for the grid by clicking on this box and choosing a color from the dialog that appears.

• Grid Transparency - Use this slider to set the transparency of the grid, relative to the background.

• Axis 0.0 color - click on the colored square to change the color of the X and Y axis (i.e. X=0.0 and Y=0.0)

All changes made in the PCA properties window take effect as soon as you click OK. To set the current settings as default click the Set Defaults button.

Additional PCA tab features

Save images (    ):

To save an image of the PCA plot, click the     button on the PCA  window tool bar. Select the location and name of the file and click Ok.

Printing (    ):

To print the PCA plot click the    button on the PCA window tool bar.

Show Principal Components (    ):

To view all the principal components of the dataset click the     button (Show Principal Components) on the PCA window tool bar, or select PCA | Show Principal Components from the PCA window menu bar. This opens a Gene Graph window showing all the principal components.

Principal Component Variance (    ):

To view the variance of the principal components (the eigenvalues) click the    button (Principal Component Variance) on the PCA window tool bar, or select PCA | Principal Component Variance from the PCA window menu bar. This brings up a Gene Graph window showing the principal component variance.

3D PCA Scatter Plots (    ):

To see the entire plot in three dimensions click the    button on the PCA window tool bar, or select PCA | Create 3D PCA Scatter Plot from the PCA window menu bar. This creates another tab in the PCA window marked “3D”. If you click on this tab you will see a 3-dimensional model of the scatter plot. Only the dots are shown. To rotate the model, click the    button (Rotate 3D Scatter Plot) and then click and drag in the window. To zoom in or out on the model click the     button (Zoom 3D Scatter Plot) and then click and drag in the window.

Save Projection and Eigenvalues (    ):

It is possible to save the projection and eigenvalues of the PCA plot to a tab-delimited file. To do so, click the    button (Save Projection and Eigenvalues) on the PCA window tool bar, or select PCA | Save Projection and Eigenvalues from the PCA window menu bar, and then choose a location and a file name in the dialog that appears. The first line of the file lists the eigenvalues. The next line lists the headers (if any are available) for the columns. Then follows the projections for each profile, using 1 line for each profile. Information in the defined info areas is included if available.

Choose Axis (    ):

In the 2D and 3D pca plots, the axis representing the 2 and 3 greatest variances respectively, are selected as default. To view the plots using other axis, press the Choose Axis (    ) button or select PCA | Set Chart Axis, and select the axis you want to use from the pull down menus.

Zoom (    ):

To zoom in on an area of interest on the PCA plot, click the Frame Method button, and then drag out a selection box. The PCA window will zoom in on the selected area. To zoom back out, click the zoom out    button. Note that zooming only works with the square selection tool (    ) .

The Three next choices are complementary. By selecting one way of handling framing of spots, you disable the two other.

Frame Contents to PCA (    ):

This option sets the zoom flag so that all framing (with square) are zoomed.

Frame Contents to Chart (    ):

This option sets the chart flag so that all spots being framed are put into a thumb diagram. This feature lets you fish out interesting areas with spots and view the corresponding elements profiles.

Toggle labels on FrameContents(    ):

This option lets you select the spots to have labels. After clicking on this button, you can either click on each spot you want labelled, or drag a lasso or frame over multiple spots.

Shadow unselected (    ):

To select certain genes, frame the area containing the genes you want to selected to chart (click    and drag out a selection box). Select the Thumbs tab and click on the new thumb. This will open a gene graph window. Genes can be selected from the list displayed to the left. Click the Shadow unselected (    ) button. The selected genes will now be clear while the unselected genes will have a shade of grey. If you go back to the PCA tab and click the Shadow unselected (    ) button once more, the selected genes will be clear, while the others will have a shade of grey. If other genes are selected, the clear and shadowed genes are updated automatically. To un-shadow unselected, simply click the Shadow unselected (    ) button again.

Repaint Component (    ):

If changes you make do not take effect immediately, press the repaint (    ) button.

Copy Clip Image to Clipboard (    ):

To copy the image in any of the tabs to clipboard, click the   button.

Show Location Thumbs

To get an instant thumbnail of the profile represented by a PCA point, select PCA | Show Location Thumb. This will bring up a small thumbnail window, which will show a thumbnail of the profile represented by the point the mouse cursor is currently over. This window has the same functionality as the Project Thumbnail window.

Show Variance:

Checking/un-checking PCA | Show Variance toggles display of variation statistics on or off.

Show Density Scale:

Checking/un-checking PCA | Show Density Scale toggles display of the Density Scale on or off, if the density map is being used.

Show tool tip box:

To get any available additional information defined in information columns of the data shown as tool tip text check the PCA | Show tool tip box. When the mouse pointer is held over a PCA point, the additional information (if any) will be shown next to it as a tool tip.

The Thumbs tab

Whenever a selection rectangle is defined that covers one or more dots (profiles) on the PCA plot, a new thumbnail is created on the Thumbs tab, containing the profiles selected.

The Thumbs tab has the same functionality as the K-Means thumbnails.

Deleting a tab (    ):

To remove a tab from the PCA window, select the tab to be removed. Then click the    button (Delete Active Tab) on the PCA window tool bar, or select Line Chart|Delete Active Tab from the PCA window menu bar. To remove the 3D scatter plot from the menu bar select PCA | Delete Active Tab instead. The PCA and Graphs tabs cannot be deleted.

Branch dataset (    ):

One additional feature that exists for the zoomed selection in the PCA window is to branch the dataset into a new node in the Project Tree. To do this, select the tab that contains the data you want to branch. Then click the    button on the PCA window tool bar, or select Line Chart | Branch Dataset from the PCA window menu bar. A new node will be added below the current one in the Project Tree labeled with the PCA symbol   .

Put in Tree (    ):

To place the entire component into the project tree click the     button, Thumbnails | Put in Tree from the PCA menu bar. This creates a new node with the symbol    in the project tree that acts as a direct shortcut to the current component.

Initiate K-Means

You can do K-Means clustering of the entire dataset based on the mean of the defined thumbnails. To this, create one or more PCA thumbnails by clicking the Frame contents to chart button (  ) and dragging out selection areas. Then select Thumbnails | Initiate K-Means. This will start K-Means analysis on the entire dataset using the mean of the thumbnails as the initialization method, and the number of cluster equal to the number of thumbnails in the PCA window.