The red underscores sometimes seen in electropherograms are regions where the capillary electrophoresis instrument has identified a peak signal that exceeds the linear range of the instrument (a saturated peak). Typically, these peaks have flat tops and smaller pull-up or pull-down peaks at the same location in the other dyes.
The significance of red underscores in electropherograms is discussed below as well as in this segment of our webinar.
The software reads saturation information from the raw data file and makes a red underscore where saturation occurs in the data. The Sample Info for Sample_1.fsa shows Saturated Area Number = 21, which means this sample has 21 peaks that exceeded the range of the CE instrument.
This is a zoomed in view of Sample_1.fsa in the green channel on the main window, showing red underscores below the trace, indicating saturated peaks. The allele labels for those peaks are yellow, notifying the analyst of a quality flag, which in this case is due to saturation repair.
The Calibration Chart for Sample_1.fsa below, also shows red underscores on the sample’s size standard electropherogram.
Sample names are listed on the left panel along with the size calibration score of 91. The score is based on how closely the size and pattern of the sample electropherogram in orange on the bottom matches the expected size standard in red on top.
Some of the expected peaks were disabled and appear in grey, while the enabled peaks appear in red. Each sample peak with a green triangle was recognized as matching the expected pattern.
Red marks below sample peaks indicate areas of saturation. Some size standard peaks have question marks indicating areas of warning for saturation and uncertainty about the peak picked, but the analyst can confirm visually that these peaks were correctly identified in this example when comparing to the expected size standard pattern.
In this example, the actual sample size peaks were properly identified and matched with the expected size pattern, but in cases where manual size calibration needs to be performed, please review our article on manually performing size calibration.