PeakTrace 6 allows users to trim traces based on 5 parameters, Q average, N count, Q count, PCR base and Clear Range trimming. These settings allow the removal of unwanted low quality data, particularly on the 3′ end.
The trim improved traces only option allows the user to decide if they want PeakTrace trimming options to act upon on all traces or just those traces basecalled by PeakTrace. This setting is useful if you want traces that were skipped short or below the good improvement threshold to be output in their original condition without trimming.
Figure 1. An example of a trace that was skipped short, with Trim improved traces only off. The low quality bases after base 304 have been removed.
Figure 2. The same trace but with trim improved traces only on. The low quality bases after bases after base 304 have been retained.
Conclusion
The trimming functions of PeakTrace are useful in removing unwanted poor quality data from the of a trace. The defaults settings of PeakTrace assume that you wish to apply the same trim settings to traces improved by PeakTrace and those not (i.e. where the original KB basecall is output). The use of trim improve traces only allows the PeakTrace improves traces to be trimmed leaving the KB basecalled traces untrimmed.
It is not advised to use the trim improved traces only setting under most circumstances.
Pitfalls With Using Trim Improved Traces Only
- Users will be giving a mix of traces some with long regions of low quality bases and some without low quality regions. Such mixing will result in users questioning the quality of their data.
- Use of the trim improved traces only setting effects the good base improvement measurement. It is very common for the KB basecaller to falsely assign high quality bases in regions of low quality. If not trimmed, these bases will be counted towards the total high quality bases in the KB input file. This can result in some PeakTrace improved traces to falling below the required good base improvement threshold. The end result is fewer traces that can be improved by PeakTrace being output.