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Example 4way plot

Create 4-way plots that show the relationships between gene (or RNA processing event) changes in two different samples (compare to 2-way plots, which show relationships between levels instead of changes). The x-axis will correspond to one comparison, and the y-axis to another. The comparisons must be pre-specified in comps.txt, but they don't have to be from the same project, or even from the same platform or genome. The magic of 4way is that the mappings between platforms and genomes will be done automatically.

Within the plot points will correspond to genes (or RNA processing events, e.g. cassette exons), and be colored according to whether they are significantly changed in the two comparisons. Genes/events changed only in the x-axis comparison are red, those changed only in the y-axis comparison are green, and those changed in both are blue. All other genes/events are plotted black.

Numbers are shown along the perimeter of the plot in different colors. They count the number of genes/events changed in one or both comparisons in different directions. The triplets of numbers at the top, right, bottom and left, count genes/events changed significantly in a single comparison, and up, unchanged or down (but below either the P-value or Fold-Change thresholds) in the other.

Each of these groups of genes can be further explored in the table browser by clicking on the adjacent action buttons.

The options for 4way are as follows:

4way options
x-axis, y-axis Three drop-down menus, which must be selected in order:
  1. Project
  2. Comparison (named comparison from comps.txt) for that project
  3. Event type: for example "genes" or "skipped exons".
P (for each axis) P-value cutoff for significance. Usually 1e-4 is used, as this roughly corresponds to 1-3 expected false positives. For skipped exons on the exon array platform, this field is instead used as a flag to decide whether or not to apply hybridization filters. P = 1 ignores these filters, 0 < P < 1 applies them (recommended).
FC (for each axis) Fold-change cutoff for significance. Default is 1.5.
Limits (x-min x-max y-max y-max) Leave these fields blank for the default behavior, which is to use a plotting window that contains all the points. Alternatively, you can specify a different boundary for any of the four sides. Use negative numbers for decreases and positive for increases. For example, x-min=-2 and x-max=5 means the plotting window will show genes/events decreased in the x-axis comparison up to 2-fold and increased in the x-axis comparison up to 5-fold.
Limit by ID Limit by ID allows you to restrict which points are plotted. Default is plot all points for which there are data, but you can supply a list

of IDs instead that you want to see, and only plot those points. These IDs might be, for example, the first column of a table browser from another comparison. In this way you can examine a gene set of interest within your data. The IDs can be uploaded or pasted (choose from drop-down menu). Pasted IDs can be separated by any whitespace. For uploaded IDs, the first line of the file is checked. If any (tab-separated) field matches /^cluster?id$/i (for example ID or clusterID), then that field is selected from the rest of the lines of the (tab-separated) file. Otherwise the file is parsed as whitespace-separated IDs.

You can also specify, using the drop-down menu, whether you want the limit applied to the x-axis or the y-axis. This only makes a difference if the two axes are from different genomes or event types.

width Width of the image in pixels
height Height of the image in pixels
Names Check the box to add the names of the genes to the plot. Often this doesn't look good, since the names tend to crowd each other out. In that case you can get the name lists by clicking the action buttons.
y=x Add the diagonal line y=x to the plot.
y=-x Add the anti-diagonal line y=-x to the plot.