basecaller
For most applications you will want to use either the default PeakTrace/KB Fallback (peaktrace/kb) or PeakTrace Basecaller (peaktrace) options. The option peaktrace/kb option will attempt to improve the basecalling using the PeakTrace Basecaller, and if this is not possible, it will fall back to processing the KB peaks. This setting will result in an improved appearance of non-peaktrace processed traces (i.e. short traces, PCR products, etc) if the clean baseline, extra baseline and/or extra normalization settings are checked.
The reltrace option outputs the raw data baseline, slightly smooth and mobility shifted with either the original KB basecall or the PeakTrace basecalled trace. The setting is useful if you wish to obtain data as close to the raw data as possible avoiding all the data manipulation that both KB and PeakTrace normally perform. This setting is useful if you wish to use the peak height data to perform downstream data analysis since the relatively peak heights in the output will match those observed in the raw data.
An alternative is to output to the original KB basecall using the abi/kb option. The abi/kb option is useful if you wish to post-process traces from the KB Basecaller.
If you are basecalling to detect polymorphisms then you may wish to use the Picket Fence option (picketfence). This applies a strong normalization to each primary peak to make them equal height while retaining the true relative heights to any sub-peaks. This can make detection of polymorphic bases simple and reliable when the traces are visually examined.
Default is peaktrace/kb.
n base threshold
The minimum quality score threshold for basecalling. Any basecalls with a quality score equal to or below this threshold value are set to N. Default is 5.
mixed peak threshold
The threshold value for secondary peaks found under the primary peak to be basecalled as mixed (degenerate) bases. This is used to detected polymorphic sites (mixed basecalling). Default is 0 (no mixed basecalling performed).
fuse trace at base
This option causes the PeakTrace output trace to be fused with original KB basecalled trace. This setting allows the better KB basecalling through dye blobs (large dye peaks) to be combined with better traces processing of PeakTrace at the 3′ end of the trace. The point at which this fusion can be set is between base 0 and 999.
It is recommended that you use as low as value as possible (1 is good). If you have significant late dye blobs (or are using the extra baseline setting) you should use a higher value to set the fuse base past the trace region affected by these large dye peaks. It is highly recommended using this setting with all KB basecalled traces.
Default is 1 (on).
fuse basecall at base
This option causes the PeakTrace basecall to be fused with the original KB basecall using the PeakTrace processed peaks. The basecall fusion can be set between base 0 and base 999. This option is an alternative to using the fuse trace option. If both fuse trace and fuse basecall are set the fuse trace setting is used and the fuse basecall option ignored. It is recommended that you use as low as value as possible (1 is normally best). If the n base threshold is 2 or higher then any N base positions in the original KB base sequence will be left as N.
Default is 0 (off).
q average trim
Sets the quality score average clipping threshold and window size. The traces are clipped using a sliding window of defined size when the average quality score falls below the set quality value. For example, if the q value is set to 10 and the window size to 20, this option will remove bases from 5′ and 3′ ends of the sequence where the average of the sliding 20 base window drops is below a quality of 10. This setting is useful for removing low quality regions from PCR product reads, etc. Trimming can be set to only the 3′ end by using the trim 3′ end only setting.
Default is q score trim of quality 12 and a window of 20).
n base trim
Sets the N base score clipping threshold and window size. The traces are clipped using a sliding window of the selected size that has more than the selected number of N bases. For example, if the N base value is set to 10 and the window size to 20, this option will remove bases from the 5′ and 3′ ends of the sequence when the sliding window of 20 bases has more than 10 N basecalls. Trimming can be set to only the 3′ end by using the trim 3′ end only setting. Default is no n base trim (0 and 20). q count trim Sets the quality score count clipping threshold, number of bases, and window size. The traces are clipped using a sliding window of the selected size that has more than the selected number of bases with a quality score below the quality score value. For example, if quality value is set to 20, number of bases is set to 4, and the window size to 20, then this option will remove bases from the 5′ and 3′ ends of the basecall sequence where a sliding window of 20 base has more than 4 basecalls with a quality score below 20. Trimming can be set to only the 3′ end by using the trim 3′ end only setting.
Default is no q count trim (0, 0 and 20).
true profile
Outputs traces using the relative true peak signal intensity. This option functions the same as the equivalent KB Basecaller function of the same name and shows the decline is signal strength. Traces that start with very strong signal and decline to very weak signal will have large peaks at the 5′ end and very small peaks at the 3′ end.
Default is unchecked (off).
skip short traces
If this option is selected then PeakTrace does not process or count usage for traces with less than the number of bases set by the skip short base option. This setting is useful if processing data sets that contain some traces derived from short PCR products.
Default is unchecked (off).
trim 3′ end only
This option modifies the trim settings so that only the 3′ end of the trace is removed by q average, n base, or q count trim settings. This setting is useful if you wish to retain the early low quality basecalls at the beginning of the trace file while removing the later low quality basecalls.
Default is checked (on).
clean baseline
This option removes trace noise from below the basecalled peaks. Secondary peak signal under the basecalled peak with a quality score greater or equal to the good quality threshold and which have been base called as an A, C, G or T is removed. Peaks with mixed basecalls or low quality bases are not cleaned. This settings results in nicer looking traces, but it does not change the basecall or quality scores.
Default is checked (on).
no peak resolution
This provides a trace output where overlapping and broad peaks are not resolved in the trace file and where the signal intensity is proportional to the raw data signal intensity. The basecalling is the same as normal PeakTrace processing.
This option is designed for the detection of polymorphisms (double peaks), or for applications where the superior basecalling of PeakTrace is desired, but where the peak intensity is proportional to the raw signal intensity.
Default is unchecked (off).
set abi limits
This setting sets the ABI Limits values as an alternative to trimming the trace when used with q average trim, n base trim, or q count trim. Rather than removing the low quality regions from the trace the peaks and bases are greyed out when the trace is viewed in Applied System’s Sequence Analysis or Sequence Scanner software. In order to use this setting the trace must have been generated by the KB Basecaller and Post Processing (PP) performed.
Default is checked (on).
Advanced Options
These options allow greater control over the PeakTrace processing and basecalling parameters, but if used unwisely they can result in suboptimal results. Always take care to understand what these setting do before changing them from their default values. More details about many of these settings and when to use them can be found in the PeakTrace In Depth article series.
good base improvement
The minimum number of high quality bases that PeakTrace must improve the basecall to generate the trace file. The allowed values can range from -999 to 999 bases. If the PeakTrace basecalling does not generate more the selected value of high quality bases then the original input trace file is output. Since PeakTrace is more conservative at calling low quality bases than KB, to correctly compare read length a negative value is required.
Default value is -20 bases.
good quality threshold
The quality score threshold for a base to be considered a high quality base. The allowed values can range from 0 to 99.
Default value is 20.
signal start peak
The location of the first peak used for basecalling. If auto is selected then largest first peak position location in the trace file is used. If first is selected the original peak location generated by the ABI Data Collection software is used. If second is selected, the most recent KB basecall peak location is used. This setting is useful if you wish to change the start point at which basecalling begins. You should only change this value from the default under unusual circumstances.
Default is auto.
skip short/pcr base
This option allows the base to be set for which traces with less than this many basecalls are considered short/PCR traces and are skipped if the skip short traces options is checked. This value can be adjusted in the range of 0 to 2000. Setting the value above 500 will turn off the skip short trace option, but it will enable traces with more than 500 bases to be PCR trimmed. This can help provide better looking traces when sequencing PCR products.
Lowering the value of this option below 500 will cause fewer traces to be considered short traces and hence more will be processed by PeakTrace.
Default is 500.
clear range trim
Sets the clear range clipping range. Bases before start or after end are removed from the 5′ and 3′ ends of the trace file and associated sequence files. For example, setting the range from 20 and 800 removes bases 0-19 and 800+ from the trace. This option is often used inappropriately limiting the possible read length and is rarely the correct trim function to use.
Default is no clear range trim (0 and 0).
extra smoothing
Allows the smoothing (blurring) of late peaks thus creating traces that more closely resemble KB output while retaining the better basecalling provided by PeakTrace. This option is purely cosmetic, but it may be of use for mollifying end users who feel that PeakTrace basecalled traces look “too good” in the later regions of the trace to be real. Use of this options enables the later peaks to be made less resolved.
The level of peak smoothing can be controlled by setting the level within the range of 1 to 9. The start base for where the smoothing is applied is controlled using base field. It is recommend that users trying this setting experiment with different level and base values, but a good place to begin testing is 3 and 850. A base value of 0 turns off extra smoothing. If you wish to apply smoothing to the entire traces then use a value of 1.
Default is no extra smoothing (3 and 0).
secondary noise peak
This option adds a secondary peak of the chosen percentage height of the primary peak under each primary peak. This option does not change the basecall, but it does make the trace appear to be more like a traces that has been generated by the KB Basecaller since the baseline will contain more noise. If the use of this setting is desired then a value in the range of 3% to 8% is optimal. It not advised to set this value over 10%.
Default is 0 (off).
extra baseline
This option applies additional baseline cleaning to the trace from the start of the trace to the skip short/pcr base value (or the fuse trace base if this is larger) to all bases with a quality score higher than the set value. This option must be used with the clean baseline setting. If significant dye blobs are present in the trace it is advisable to use fuse trace option with a large value (i.e. 300) to avoid converting dyeblob peaks into peaks that appear to be actual peaks.
If the trace contains mixed bases (polymorphisms) it is strongly advised to used mixed basecalling (i.e. use a mixed peak threshold greater than 0). This option does not affect the base or quality call, but it does improve the appearance of the traces.
The ideal value to use for this is setting will depend on user needs. To remove dyeblobs use a low value (under 10), for more general use a value of 10 to 15 is likely to be better.
Default is 0 (off).
force processing
Forces the processing of trace files that are normally rejected by PeakTrace due to poor quality. Use this option with care as force processing can result in the generation of corrupt and/or nonsense files.
Default is unchecked (off).
trim improved traces only
Applies any base trimming to the PeakTrace improved traces only and not the original KB basecalled trace. It is rare to want to limit the trimming to the PeakTrace basecalled traces only so be cautious changing this from the default.
Default unchecked (off).
stealth mode
Hides that the trace was basecalled by PeakTrace. Both the .phd.1 and .scf file will record the trace processor and basecaller are KB 1.4.
Default is unchecked (off).
noise raw data
This replaces the raw data channels with random noise signal. This can be used to prevent re-basecalling of the resulting .ab1 file by the KB Basecaller.
Default is unchecked (off).
simple mixed basecall
This option changes the mixed basecalling from the standard (strict) mixed basecall (mixed peak threshold) to a simplified mixed basecall based on the secondary peak signal heights at the basecall locations in the processed channels. This setting will ensure that any secondary signal above the mixed peak threshold level is called as a mixed base even if it does not look like a true peak. This is similar to the way KB calls mixed peaks.
This simple mixed basecalling is inferior to the standard mixed basecalling and it is advised that this setting is not used unless needed as it will call more false mixed bases.
Default is unchecked (off).
extra normalization
This option performs additional normalization on the peak heights to ensure that the peak heights are of near equal in height all the way down the trace file. This option will hide significant declines in signal strength resulting from poor sequencing conditions so its use is cautioned.
Default is unchecked (off).
use edited basecalls
This option allows the use of the edited rather than the original basecalls contained within the KB basecalled trace file for any function (e.g. fuse trace, fuse basecall, or any of the trim functions). This setting can be useful if you are using PeakTrace after a trace has been manually edited.
Default is unchecked (use original basecall).