One of the new features introduced in PeakTrace 6.10 is the “picket fence” basecalling option. This option is normal PeakTrace basecalling with an extreme peak normalisation applied that makes all the trace peaks approximately the same height. This output can be very useful if you wish to post-process the trace file to detect and quantify polymorphic regions.
Figure 1 shows an example KB basecalled trace containing a polymorphic region resulting in mixed base calls. The primary peaks vary by more than 4-fold in height making the quantification of the relative heights of the mixed positions more complex.
Figure 1. KB basecalled trace. Mixed peak threshold 30%.
Figure 2 shows the same trace file basecalled using the standard PeakTrace basecalling option. A similar primary peak height variation can be seen with PeakTrace basecalled trace as is observed with the KB trace which is a reflection of the underlying raw peak heights.
Figure 2. PeakTrace basecalled trace. Mixed peak threshold 30%.
Figure 3 shows the same trace basecalled using the “Picket Fence” basecalling option. The peaks display an extreme normalisation making it easier to quantify the secondary peak heights at each of the polymorphic positions.
The picket fence basecaller is not recommended for normal basecalling applications as it creates a very artificial looking trace file, however, the very even primary peak heights can make identification and quantification of polymorphic sites more robust. The picket fence basecaller is available in Auto PeakTrace 6.10 and higher, PeakTrace 6.10 and higher, and Auto PeakTrace RP 6.10 and higher.