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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.
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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 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.
After you have set the threshold levels to yield results, you click the Make Track button. After an annotation track has been created with Make Track, it is frozen and is no longer linked to changes in the Graph Thresholds window. But you are free However, you can continue to modify the criteria in the Graph Thresholds window, altering the threshold results for the same graph track and then create another annotation track.
To capture the threshold bars and convert them to annotation tracks:
- Click to select the graph
- Click the Make Track button.
A snapshot of the current thresholded graph will be created as a new annotation track. This track will be given a default label containing all of the relevant parameters, which you can later change if desired.
To permanently capture the threshold snapshot for later use or sharing:
- Right-click the track and choose Save Annotations.
- Choose one of the file formats.
- Name your file. Be sure to include the correct filename extension.
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 annotationsAn example name is
- threshold, 60 to offsets: (+12, +13), max_gap=20, min_run=30, graph: depth: HotI2T1
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.