Identification of sharp sequencing trace spikes
- The trace file contains narrow spikes (from 2 to 9 data points) in the raw data channel that can have intensities as high as 32,000 (detector limit).
- The spike is in all four channels at the same position.
- The sharp peaks are normally higher than the surrounding sequence peaks.
- The sharp peaks are more common towards the end of the trace than the beginning, but they can occur anywhere in the trace.
Figure 1. Example of a sharp signal spike
Figure 2. Raw channel data at the same location as Figure 1. Note that it reaches over 3000 in intensity.
Cause of sharp trace signal spikes
- The precise cause of sharp signal spikes is a bit of a mystery. The best theory is these spikes are caused by small gas (air) bubbles forming in the capillaries during electrophoreses. These bubbles refracts the laser light onto the CCD camera as they pass by the detector zone. The frequency of sharp spikes seems to be both operator and machine dependent.
Solving sharp trace signal spikes
- The best solution is to purge the buffers and load a fresh polymer matrix. Avoiding excessive shaking of the buffers and polymers before loading on to the instrument seems to help.
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