One of the new features introduced in PeakTrace 6.40 was secondary noise peaks. The setting is unusual in that it actually make the traces appear to be of lower quality than the result normally provided by PeakTrace. This setting introduces artificial secondary peaks under the primary peak proportional in height to the percentage value entered and the height of the primary peak. For example, using a secondary noise peaks value of 5 adds secondary peaks that are 5% of the primary peak heights under every primary peak in the trace.
Adding secondary noise peaks can be useful if you wish to give your end users a trace file that looks much closer to that which would be output by the KB Basecaller. Figures 1 to 3 show examples of how the secondary noise peaks option works. Figure 1 shows an original KB Basecalled trace at ~base 400; Figure 2 shows the same trace after PeakTrace basecalling with a secondary noise peaks setting of 0 (i.e. off); Figure 3 shows the same trace, but using a secondary noise peaks setting of 5 (i.e. 5%).
Figure 1. KB basecalled trace.
Figure 2. PeakTrace basecalled trace with default processing values.
Figure 3. PeakTrace basecalled trace with a secondary noise peaks setting of 5.
While it is immediately obvious that the standard PeakTrace basecalling (Figure 2) is far superior to that provided by KB (Figure 1), some users are concerned that the default PeakTrace basecalled traces look “too good” to be real. While we think this is an unnecessary concern (after all nobody complains that their dinner tastes “too good” and they wished that it tasted worse), we do understand that some of our customers want to provide their end users with the superior basecalling of PeakTrace with a trace that looks very similar to what KB generates. The secondary noise peaks setting provides a simple way of degrading the trace output so that PeakTrace basecalled traces look like KB basecalled traces, while still providing the same base and quality score improvements that PeakTrace offers.
It is advisable to not use a secondary noise peaks value over 10. Using a large value will create a trace with a very poor appearance and end users may mistake the secondary peaks as true mixed peaks. This is one setting to use with caution.