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Nucleics

Update to the Free PeakTrace Online Settings

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atp_rp_options_free

With the release of PeakTrace 6.40 we have updated the setting for the free version of PeakTrace (PeakTrace Online) to make optimal use of the improvements incorporated into this version. The new settings are now

-t abi -ft 300 -c -40 -wq 12 20 -xb 7 -cb yes -bc pk -ts no -xs 1000 2 -sm 700

These values correspond to the Auto PeakTrace and Auto PeakTrace RP Option settings:

  • fuse trace at base: 300
  • good base improvement: −40 bases
  • q average trim: 12 threshold; 20 base window
  • clean baseline: yes
  • extra baseline: 7
  • basecaller: peaktrace/kb (PeakTrace Basecalling with KB fallback)
  • trim 3’ end only: yes
  • extra smoothing: level 2; start base 1000
  • skip short/pcr base: 700

These settings are not suitable if you are trying to detect polymorphic sites (i.e. mixed peaks). For this you need to use a mixed peak threshold above the default 0.

The PeakTrace default values are usually the best place to start, but you should always trial the various options to find what works best for your needs.  If you would like further advice on the best settings to use with your sequencing data, please don’t hesitate to contact us  @ {This email is obscured. Your must have javascript enabled to see it}.

Auto PeakTrace RP 6.41 Released

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Comparison between PeakTrace and KB basecallersWe have released Auto PeakTrace RP 6.41. This is a minor update, but with a number of important changes to the internal modules that improve the performance and basecalling quality. The full list of changes include.

  • Improvements to the basecalling of traces containing mixed bases.
  • Improvements to clean baseline and extra baseline processing of traces.
  • Improved handling of traces that are basecalled with kb or peaktrace/kb options.
  • Improvements to mixed basecalling module.
  • Reduced memory requirements and increased processing speed.

Auto PeakTrace RP 6.41 can be downloaded from our PeakTrace RP Downloads page for both Windows (XP, Windows 7, Windows 8 and Windows 10) and MacOS X (10.6+). It is also available via the in-app update function. If you do not have the password to access the downloads page please contact us @ {This email is obscured. Your must have javascript enabled to see it} or contact your local supplier.

If you are not a current user of Auto PeakTrace RP 6 then please visit the Auto PeakTrace 6 RP registration page.

Auto PeakTrace 6.40 Released

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Auto PeakTrace 6.40 Options Window

Auto PeakTrace 6.40 Options Window.

We have released Auto PeakTrace 6.40 and PeakTrace 6.40 for Linux x86_64. This is a major release that improves the processing, basecalling, and appearance of traces. We have updated the Options menu of Auto PeakTrace 6 making it easier to use. We have also changed the the default settings to make the best use of the new features incorporated into this release and the Users Manual has been updated and improved. Finally, PeakTrace 6.40 has a new feature which allows the ABI Limits values to be set by PeakTrace instead of performing hard trimming of the trace files. The full list of changes

  • New Feature. Set ABI Limits option.
  • New Feature. Improved Options layout and default settings.
  • Improvements to the extra baseline setting to allow user control of the threshold for secondary peak removal.
  • Improvements to the clean baseline setting to allow user control of the threshold for removing trace noise.
  • Improved processing of traces generated using BigDye™ 1.1 chemistry.
  • Updated Help/Users manual.
  • Initial support of the NimaGen BrilliantDye™ Terminator chemistry.
  • Bug fixes and improvements.

The update is highly recommend for all user of Auto PeakTrace 6 or PeakTrace 6.  Both the Linux and Windows version of PeakTrace 6.40 can be downloaded from the PeakTrace:Box Downloads page. It is also available via the in-app update function within Auto PeakTrace 6. If you do not have the password to access the downloads page please contact us @ {This email is obscured. Your must have javascript enabled to see it}.

Auto PeakTrace RP 6.40 Released

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Auto PeakTrace RP 6.40 Options Menu

Auto PeakTrace RP 6.40 Options Menu

We have released Auto PeakTrace RP 6.40. The is a major update that significant improves trace quality output and appearance. We have changed the Options menu to make Auto PeakTrace RP easier to use and updated the default settings to make best use of the new settings. In addition, we have improved the help manual and added a new feature which allows the ABI Limits values to be set by PeakTrace instead of hard trimming. The full list of changes include.

  • New Feature. Set ABI Limits.
  • New Feature. Improved Options layout and default settings.
  • Improvements to the extra baseline setting to allow user control of the threshold for secondary peak removal.
  • Improvements to the clean baseline setting.
  • Improved processing of traces generated using BigDye 1.1 chemistry.
  • Updated Help manual.
  • Initial support of the NimaGen BrilliantDye™ Terminator chemistry.
  • Bug fix for the abi+kb output option.

This update is highly recommend for all user of Auto PeakTrace RP 6.30 or earlier. If you had been using the extra baseline setting with Auto PeakTrace 6.30 then you must upgrade to this version as the 6.30 version of extra baseline will no longer work.

Auto PeakTrace 6.40 can be downloaded from the PeakTrace RP Downloads page for both Windows (XP, Windows 7, Windows 8 and Windows 10) and MacOS X (10.6+). It is available via the in-app update function. If you do not have the password to access the downloads page please contact us @ {This email is obscured. Your must have javascript enabled to see it}.

If you are not a current user of Auto PeakTrace RP 6 then please visit the Auto PeakTrace 6 RP registration page.

In Depth: Extra Baseline

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One of the new features of PeakTrace 6.30 is the extra baseline setting. This setting further improves the appearance of trace files by removing non-base peaks such as dye-blobs and baseline noise. It does not affect the base or quality call of the output file. This setting is best illustrated with an example trace.

Figure 1 shows an example trace that has been basecalled with the KB Basecaller™. The trace contains significant dye-blobs and noise peaks under the primary peaks. The appearance of the trace is not ideal.

Figure 1. No baseline cleaning.

Figure 1. KB basecalling.

Figure 2 shows the same trace and region basecalled with PeakTrace 6.30 using the clean baseline setting. Much of the noise peaks have been removed, but the dye-blob region is still poor in appearance.

clean_baseline

Figure 2. PeakTrace basecalling using clean baseline.

Figure 3 shows the same trace and regions as Figures 1 & 2, but using PeakTrace 6.30 and the extra baseline setting. The dyeblob region and noise peak signal has been removed from the trace. Note the DNA sequence and quality scores are identical in all three traces.

extra baseline processing

Figure 3. PeakTrace basecalling using extra baseline.

Pitfalls with using Extra Baseline

The extra baseline setting is a very powerful tool for improving the appearance of the trace files, but like all powerful tools it can prove problematic if abused. The three major issues to watch out for are:

  1. Applying extra baseline to traces with polymorphic bases (double peaks) and not using mixed basecalling.  If you are going to use extra baseline on traces with mixed bases then it is imperative that you use the mixed basecall setting (i.e. setting the mixed peak threshold to great than 0) in both KB and PeakTrace.  !! Failure to do this will result in the removal of the mixed peaks !!
  2. Large dyeblobs can cause problems of assigning the wrong peaks in the dyeblob region. The quality scores of these peaks will be low, but to an end user it will appear that the peaks are excellent and they may choose to ignore that the region is poor quality. The best solution is to use a large fuse trace or fuse basecall value (e.g. 250 to 300). This will minimize the chance that dyeblobs are turned into incorrect peaks.
  3. Using a large (> 1000) skip short/pcr base value can result in unnatural looking traces. The extra baseline setting is applied to the trace up to the skip short/pcr base. If it is required that a large skip short/pcr base value be used, then it is recommended that extra smoothing is also used. This will ensure the trace looks far more like a normal KB basecalled trace.

Update

The extra baseline setting has been changed in PeakTrace 6.40 to allow more user control over the threshold at which it is applied. Users now can control the quality score threshold below which extra baselining is not applied. For example, if the extra baseline setting is set to 15 only bases 15 or greater will have the additional baseline cleaning applied. This can avoid turning poor quality traces into traces that appear to be good, as well as avoiding the removal of mixed peak locations.

 

Auto PeakTrace 6.30 Released

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Auto PeakTrace 6.30 Options Window

Auto PeakTrace 6.30 Options Window

We have released Auto PeakTrace 6.30. This is a major release incorporating two new features for improving trace appearance – extra baseline and secondary noise peak. In addition, a number of the underlying algorithms used in the PeakTrace basecalling process have been improved further enhancing trace processing quality and read length.

The full list of changes in Auto PeakTrace 6.30

  • New Feature. Extra baseline option for improved appearance of trace files.
  • New Feature. Secondary noise peak option to add peak noise to the traces.
  • Auto PeakTrace 6.30 requires CodeMeter runtime 6.20 or higher.
  • Improved trace processing quality and speed.
  • Unsupported run types now output .seq and .phd.1 files based on the KB basecall if these file types are requested.
  • Trace types which do not contain quality scores can now be output in ab1 format without quality scores if either clean baseline, extra baseline, or extra normalization is selected.
  • Additional check that the archive folder can be created when using the archive traces option.
  • Improved handling of traces processing when manually starting and stopping processing.
  • If the N base threshold is set to 1 or 0 and the fuse basecall value is greater than 0, then any N bases from the original KB basecall will be set to a quality score of 2.
  • Bug fixes and enhancements.

The release is recommended for all users of Auto PeakTrace earlier than 6.12 and is an optional update if you are running Auto PeakTrace 6.20. If you are using a release before 6.20 please contact us @ {This email is obscured. Your must have javascript enabled to see it} (or your local supplier) before installing as we will need to be provided with the PeakTrace Serial Number before you can use this update.

This update is available from the PeakTrace:Box Downloads page or if you are running Auto PeakTrace 6.20 it will be available from the built in update function of Auto PeakTrace.

PeakTrace 6.30 Linux x86_64 Released

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extra baseline processingWe have released PeakTrace 6.30 for x86_64 Redhat/CentOS 6.7. This update incorporates a number of major changes that improve trace processing and output quality. The full list of changes

  • New Feature. Extra baseline option (-xb) for improved appearance of trace files.
  • New Feature. Secondary noise peak option (-nsp) to add peak noise to the traces.
  • Improved trace processing quality and speed.
  • Unsupported run types now output .seq and .phd.1 files based on the KB basecall if these file types are requested.
  • Trace types which do not contain quality scores can now be output in ab1 format (without quality scores) if either clean baseline, extra baseline, or extra normalization is selected.
  • If the N base threshold is set to 1 or 0 and the fuse basecall value is greater than 0, then any N bases from the original KB basecall will be set a quality score of 2.
  • Requires CodeMeter runtime 6.20 or higher (provided).
  • Bug fixes and enhancements.

PeakTrace 6.30 can be downloaded from the PeakTrace:Box Downloads page. If you do not have the password to access the PeakTrace:Box Downloads page please contact us @ {This email is obscured. Your must have javascript enabled to see it} or contact your local supplier directly. If you are current running a version of PeakTrace from before 6.00 you may need a license upgrade.

We will be releasing Auto PeakTrace 6.30 early next week as soon as the final testing is complete.

New Settings for the free version of PeakTrace Basecaller

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With the release of Auto PeakTrace RP 6.30 we have updated the basecalling parameters used by the free version of PeakTrace to take advantage of the new options available within PeakTrace 6.30. The PeakTrace command line options settings now used are:

-t abi -ft 300 -c -40 -wq 12 20 -xb yes -cb yes -bc pk -ts no -xs 1000 2 -sm 700

These values correspond to the following Auto PeakTrace and Auto PeakTrace RP Option settings.

peaktrace_online

The Auto PeakTrace setting corresponding to PeakTrace Online settings.

These settings are designed to optimise the read length and appearance of the trace, but not the detection of polymorphic sites. If you wish to detect polymorphic sites (mixed bases) raise the mixed peak threshold above 25%.

If you would like further advice on the best PeakTrace settings to use with your sequencing data, please don’t hesitate to contact us  @ {This email is obscured. Your must have javascript enabled to see it}.

In Depth: QualTrace III Trace Reports

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QualTrace III Options menuThe QualTrace III module in PeakTrace 6 can generate text based Trace Report files on each trace file basecalled by PeakTrace. These files can be supplied to final customers to help them understand why their trace is not to the quality level they might wish and also what they might do to correct the problems.

The Trace Report files can be generated with increasing levels of report detail, depending on the user needs. The report files are named after the trace file with “_report.txt” added (e.g. if the trace file name is “trace_abc.ab1” then the trace report file will be named “trace_abc_report.txt”) and saved to the same folder as the output trace file.

The Trace Reports can be generated with increasing levels of detail as desired. Trace Reports are only available with the PeakTrace:Box version of the PeakTrace Basecaller and are not available with the Auto PeakTrace RP system. The details of what each output means are described in the QualTrace III User manual.

Auto PeakTrace 6.50 introduced QualTrace III only trace reports, allowing them to function independently of PeakTrace. When qualtrace is selected as the basecaller, all references to PeakTrace (e.g. PeakTrace class, PT Good Bases, and Improvement) are omitted from the trace report. In addition, the PeakTrace settings section will not be included in the full report.

The four Trace Report options and outputs are:

No Report

This option prevents the output of a trace report. This is the default value.

Brief

Outputs a short description of the most important QualTrace III analysis results. An example is shown below.

SUMMARY
Trace File: ABI_3130_1200_B.ab1
PeakTrace Class: Success
Total Bases: 1367
KB Good Bases: 1333
PT Good Bases: 1340
Improvement: 0.5%
Date: 2015/08/11 10:55:00
Trace Score: 50
Contiguous Read Length: 1344

Standard

Outputs a summary and detailed description of the important QualTrace III analysis results. An example is shown below.

SUMMARY
Trace File: ABI_3130_1200_B.ab1
PeakTrace Class: Success
Total Bases: 1367
KB Good Bases: 1333
PT Good Bases: 1340
Improvement: 0.5%
Date: 2015/08/11 10:57:38
DETAILS
QualTrace Class: Excellent trace
Average Peak Spacing: 14.41
Run Time: 9000 sec.
Total Scans: 17960
Detector Overload: 0
Trace Score: 50
Contiguous Read Length: 1344
Dye Signal: 0.94
Bubble Spike(s): 0
Void Peak(s): 0
First Peak at Scan: 1276
Early Signal to Noise: 80
Mid Signal to Noise: 64
Late Signal to Noise: 46
Signal at Noise Level at Base: 1406
InDel(s): 0
Early Mixed Peaks: 0.00
Late Mixed Peaks:0.00
Peak Width at Base 700: 13.29

Full

Outputs the summary and full details of the QualTrace III analysis in text form including the PeakTrace and QualTrace III settings used for the analysis. An example is shown below.

SUMMARY
Trace File: ABI_3130_1200_B.ab1
PeakTrace Class: Success
PeakTrace Code: 0
Total Bases: 1367
KB Good Bases: 1333
PT Good Bases: 1340
Improvement: 0.5%
Date: 2015/08/11 11:00:21
DETAILS
QualTrace Class: Excellent trace
QualTrace Code: 40
Sequencer: 3730-16112-027-16112-027
Run Module: XLRSeq50_POP7_1
Run: Run_3730-16112-027_2007-03-17_16-00_0297
Run Date: 2007/03/17 17:00:52
Original Basecaller: PT 5.51
Capillary Number: 23
Capillary Length: 50
Polymer Lot: 0701015
Tray: 343
Tray Well: B5
Sample ID: 58e5d004d3f311ebaabf000d56d7a0f8
Sample Name: 23997
Sample Comment: <ID:23997>
Sample Owner: Daniel_Tillett
Sequencer User: Daniel_Tillett
Run Temperature Set: 60 deg.C
Run Voltage Set: 6.10 kV
Scan Rate: 0.25 sec.
Measured Run Voltage: 6.0 kV
Measured Run Current: 143.0 mA
Measured Run Power: 25.0 mW
Measured Run Temperature: 60.0 deg.C
Injection Voltage: 15.0 kV
Injection Time: 15 sec.
Average Peak Spacing: 14.41
Run Time: 9000 sec.
Total Scans: 17960
Detector Overload: 0
Trace Score: 50
Contiguous Read Length: 1344
Average Detector Noise: 5.21
Spectral Calibration: 0.08
Dye Signal: 0.94
Bubble Spike(s): 0
Void Peak(s): 0
First Peak at Scan: 1276
Early Signal to Noise: 80
Mid Signal to Noise: 64
Late Signal to Noise: 46
Input Total Bases: 1406
Signal at Noise Level at Base: 1406
Quality Score Threshold: 20
InDel(s): 0
Early Mixed Peaks: 0.00
Late Mixed Peaks: 0.00
Peak Width at Base 700: 13.29

PEAKTRACE SETTINGS
Trace File Output: ab1
Seq File Output: abi
.phd.1 file output: no phd
Good Base Improvement: -20
Good Quality Threshold: 20
N Base Threshold: 5
Mixed Peak Threshold: 0
Signal Start Peak: auto
Basecaller: peaktrace
Fuse Trace at Base: 20
Q Average Trim: 12 20
Force Processing: no
True Profile: no
Skip Short Traces: no
Trim Improved Traces: yes
Stealth Mode: no
Noise Raw Data: no
No Peak Resolution: no
Clean Baseline: no
Extra Normalization: no

QUALTRACE SETTINGS
Delayed First Peak: 133%
Crosstalk Threshold: 15%
Signal to Noise Base: 700
Signal to Noise Threshold: 4
Early Signal Base: 80
Mid Signal Base: 64
Late Signal Base: 46
Poor Resolution Threshold: 300%
Poor Resolution Base: 700
Quality Threshold: 20
Contiguous Readlength: 20 20
Mixed Peak Threshold: 50%
Mixed Trace Threshold: 40%
Early Mixed Base Start: 50
Early Mixed Base End: 250
Late Mixed Base Start: 500
Late Mixed Base End: 700
Bubble Spike Threshold: 200%
Void Peak Threshold: 300%

Why Keep PeakTrace Up-To-Date?

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Auto PeakTrace Options MenuOne thing we have noticed is many people are running old version of PeakTrace. While it is in general a good idea to use software you are familiar with, with PeakTrace it is best to keep up to date as much as possible. there are a number of very good reasons to use the latest version

  1. PeakTrace will stop working correctly approximately 18 months after it is first release. It will still run, but the quality of the results will be very poor. Anyone running a version of PeakTrace from before 5.70 should upgrade today.
  2. PeakTrace is actively being developed and improved. Unlike most Sanger sequencing software companies, Nucleics is continuing to invest significant resources in improving PeakTrace. By running the latest version you get access to all these improvements. Some of the great new feature of PeakTrace 6 are described in the PeakTrace 6 In Depth articles.
  3. Bugs are only fixed in the latest version. If you run into an issue or bug with an older version of PeakTrace then there is a good chance it has been fixed in a later release.  If the issue is new then we will be able to fix in the next release. If the bug is specific to an older release then it will not be fixed.
  4. It is free. If you have a current license you can upgrade at anytime at no cost.