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Introduction
A common analysis step is to determine meaningful regions on a sequence based on graph values being above or below a certain threshold. To screen out non-meaningful values and to view meaningful features of a graph as annotations, use the thresholding feature in IGB. Thresholding displays graph features as annotation-like bars in locations where the graph value at the coordinate meets your defined threshold.
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The Offsets for Thresholded Regions feature is designed specifically for working with tiling array data. Captured data from tiling array probes should usually be shifted for display. Since the probes are 25 base pairs long, but the x-coordinates given represent the starting coordinate, you should shift the threshold data so that it starts at 12 base pairs past the given beginning, and ends at 13 base pairs past the beginning. This is the default placement of all graph threshold bars; if you are viewing typical tiling array data, IGB default is set to the correct parameters. If these offsets are not correct for the data you are analyzing, you should change them.
When you view non-tiling array data in IGB, you should adjust the offset to 0 (set Start to 0 and End to 1) to correctly align the threshold bars with their actual coordinates.
Control gaps
Experimental data can be noisy. If you are looking for general trends and want to ignore small local variations, you can use the Max Gap and Min run settings. Slide the sliders to adjust, or enter values into the boxes for each parameter:
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Min run: minimum number of bases in a row that must meet the threshold before an annotation bar will appear. For example, by default the Min run is 30 base pairs, so a sequence of 29 base pairs that meet the threshold will not be marked by an annotation bar. Again if you species/ gene models typically have very short exons, you may need to reduce this number. On the other hand, if your species has a minimum size for exon length to be meaningful, you can adjust this higher.
Make an Annotation Track of the Threshold Results
As shown above, while you are working with threshold values, the 'bars' appear in the same track as the graph itself. However, you may wish to record the threshold results before changing values again; you may wish to work with the thresholding results as a separate track; you may with wish to save the results to share with others. For all of these reasons, you can create an annotation (NOT a graph) track of the thresholding results. After you have captured threshold "annotations" from a graph or other set of data, you can examine these "annotations" using all of the tools in IGB for working with and comparing annotations.
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This track will be given a default label containing all of the relevant parameters, which you can later change if desired. An example name is
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The name of the new track will always start with 'threshold' and end with the name of the graph track that was used (in this case, the graph was a depth graph made from a .bam track called HotI2T1). The value '60 to infinity' shows that the threshold level was set to 60 and looked at > thresh. The offsets were NOT corrected as they should have been, and are set to the tiling array values. Finally, the max gap and min run values are recorded.