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How to Install PeakTrace License Update

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A common question is how to install the PeakTrace License Update File so that the license on your USB key is current. It is very simple process.

1. When you receive the update file (most likely by email) save it in a suitable location that you can access from the computer with the USB key is installed. Note that the update file will have a green down arrow (see circled file in the below figure) and the file name extension will end in a U (e.g. 2-1862648.WibuCmRaU)

Step 9

2. Double click on the update file to install it. Press Yes.

Step 10

3. If the update installs correctly then you should see the below window. Press OK to finish.

Step 11

4. You can now check your new License and the days and units remaining on the bottom left hand side of the Options windows of Auto PeakTrace.
license

How to Update the PeakTrace USB Key Time

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The PeakTrace USB Key contains an independent clock that must be kept in alignment with the system time of your computer. Normally this clock remains accurate for many months, but after a license or firmware upgrade it may need to be updated. The process to do this is simple:

1. Open the CodeMeter Control Center from Run > All Programs > CodeMeter. Please make sure that the USB Key supplied by Nucleics is plugged into your computer. This computer must have internet access. If the computer doesn’t have internet access then you can move the USB key to a computer with access (and CodeMeter installed) and do the time update on that computer. The updated time will be retained when you move the USB key back to the original computer

UpdateTime_1

2. Click on the Process menu option.

UpdateTime_2

3. Click on Update Time Certificates.

UpdateTime_3

4. Once the USB clock is successful updated the above window will appear.

Linux & Windows command line

From the command line (linux & windows) you can also use the following command to update the time:

cmu --time-update

How to Upgrade to Auto PeakTrace 6

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Here are steps required to upgrade Auto PeakTrace. The links below will take you to the specific page(s) that explain how to do each step.

  1. Update your CodeMeter Runtime to the latest version (7.60a as of May 2023).
  2. Download the Auto PeakTrace 6 Installer from the Nucleics PeakTrace:Box Downloads page and install Auto PeakTrace 6. You will need a password to access the downloads page which will be supplied to you by Nucleics or your local supplier.
  3. You may need to reactivate your PeakTrace license online. If you need to do this you will be prompted to enter your PeakTrace Serial Number. The PeakTrace Serial Number is a 20 character long value broken into 5 blocks of 4 characters (i.e. XXXX-XXXX-XXXX-XXXX-XXXX). It is not the same as the PeakTrace License Code which is accessed from the Options window. This will have been supplied by Nucleics (or your local supplier) in the past. You should not need to do this step for versions 6.96 and higher.
  4. You should now be able to start and run Auto PeakTrace 6. It is a good idea to read through the help manual as there will likely have been many changes made and new featured added.

Troubleshooting

  • If you get the error message “Auto PeakTrace 6 Key Invalid” then try updating the USB Key Time and the system time on the computer. These two times must be in agreement.
  • Try restarting the computer and check again.
  • Make sure that your operating system is one of those that are supported (Windows XP, Windows 7, Windows 8 or Windows 10 or Redhat/CentOS 6.9 or higher). Auto PeakTrace 6 has not been tested on Windows 11, but it should work.
  • If you are installing on Windows XP then the current CodeMeter runtime will not install. We provide an older version of CodeMeter (6.80) on the PeakTrace:Box Downloads page.
  • Make sure that you are not trying to run Auto PeakTrace 6 on a virtual machine. Doing so will lock your USB key and delete your license.
  • If Auto PeakTrace 6 won’t start at all, you may be missing one of the required Windows runtime libraries. You can download the missing Windows libraries from the Microsoft website.
  • If all these efforts fail, please email a cmDust file to Nucleics (or your local supplier) along with a newly generated PeakTrace Licence Request file. This will allow us to diagnose the problem with your license or system.

How to update the CodeMeter Runtime

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All version of Auto PeakTrace above 5.70 require the latest CodeMeter Runtime (6.90 as of September 2019). To update you first need to check which version of the CodeMeter Runtime you have installed on your computer. To do this:

1. Open the CodeMeter Control Center from Run > All Programs > CodeMeter. Please make sure that the USB Key supplied by Nucleics is plugged into your computer.

About

2. Click on the Help menu and then on About.

about_runtime

3. The version number will be shown on the About window. If you runtime version is less than 6.80 then you need to update the CodeMeter Runtime. The latest CodeMeter runtime can be downloaded from Wibu. If you are running PeakTrace on Windows XP you will need to use CodeMeter 6.80. This version can be downloaded from the PeakTrace:Box downloads page.

4. Run the Codemeter Runtime Installer following the prompts. After it finishes restart your computer.

How to Update the PeakTrace License on Linux

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A question we occasionally get asked is how to generate and update the PeakTrace license from the command line when using the Linux version of PeakTrace. The step–by–step process involves

1. Open a shell window with the USB Key plugged in and enter:

cmu --list

This command will list the serial number of the CmContainer which you will need to generate the PeakTrace License Request file.

cmu_list2. Create the license request file using:

cmu --context 101945 --file [serial number].WibuCmRaC

cmu_create_file3. Email this file to Nucleics (or your local supplier).

4. You will be returned an updated license file which can be imported into the PeakTrace USB Key using:

cmu --import --file [serial number].WibuCmRaU

cmu_import

5. Update the time on the key using:

cmu –time-update

In Depth: Mixed Peak Threshold over 100%

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One of the less obvious setting in PeakTrace is using a mixed peak threshold value over 100%. The mixed peak threshold controls the value at which as basecall with a secondary peak is called as a mixed base. For example, if the mixed peak threshold is set to 40% then any peaks with a secondary peak 40% or greater than the height of the primary peak will be called as a mixed (degenerate) base.

A secondary function of the mixed peak threshold value is controlling the removal of secondary peaks smaller than the threshold value when using the clean baseline and extra baseline options. If a mixed peak threshold value of 20% is used along with extra baseline and clean baseline then those secondary peaks smaller than 20% of the primary peak will be removed from the trace. Any peaks with a secondary peak higher then the threshold value will be called as a mixed base.

If it is desired to keep the secondary peaks above a certain size, but not call the peaks as mixed/degenerate then using a mixed peak threshold over 100% will enable both the minor secondary peaks to be removed and the major secondary peaks to be kept while the primary peak is called as the base.

Figure 1 shows an example of a trace with a significant amount of noise, but also some true mixed peaks. These can be very difficult to detect in noisy traces as the processing KB performs on the raw data can remove or change the height of both true peaks and noise peaks.

Figure 1. KB Basecaller

Figure 1. KB Basecaller™ processed region

Figure 2 shows the same peak region but processed with PeakTrace no peak resolution processing. This processing mode outputs the raw data with minimal processing (signal baseline and dye mobility shift correction) enabling the true peak data to be examined. A complex pattern of secondary peaks is observed in Figure 2 that are missing from the KB trace.

Raw data (no peak resolution).

Figure 2. Raw data (no peak resolution).

Figure 3 shows the same region processed with standard PeakTrace basecalling and processing. While the secondary peaks are better separated from the noise peaks and have heights that correspond to the peak heights seen in the raw data, many of these secondary peaks are small and not significant mixed bases.

Standard PeakTrace processing and basecalling.

Figure 3. Standard PeakTrace processing and basecalling.

Figure 4 shows the same trace region but with the mixed peak threshold set to 40% with clean baseline and extra baseline used. While only the large secondary peaks are retained the basecall contains degenerate/mixed basecalls.

PeakTrace mixed basecalling at 40% with clean baseline and extra baseline.

Figure 4. PeakTrace with mixed basecalling at 40% with clean baseline and extra baseline.

Figure 5 shows the same region but using a mixed peak threshold of 140% along with clean baseline and extra baseline. The trace looks the same the 40% trace (i.e. only the major peaks are retained), but the basecall does not contain any degenerate/mixed basecalls. The basecall at the degenerate position is set to the primary peak call.

Figure 5. PeakTrace with mixed basecalling at 140% with clean baseline and extra baseline.

 

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}.

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%

In Depth: Mixed Basecalling and Simple Mixed Basecalling

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One of the major internal changes within Auto PeakTrace 6 is an improvement to how mixed basecalling (detection of polymorphic sites)  is performed. PeakTrace 5 used a similar algorithm to the KB Basecaller. Both called a base as mixed if the secondary signal beneath the primary peak of the processed data exceeds the mixed peak threshold. For example, if the mixed peak threshold is set to 30% then any peak with secondary signal greater than 30% of the primary peak height is called as a mixed base. Mixed basecalling is switched on by using a mixed peak threshold of greater than 0.

Figure 1. Polymorphic region basecalled with KB.

Figure 1. Polymorphic region basecalled with KB with a mixed peak threshold of 30%

While this approach closely replicates how KB works, it does suffer from two major issues. First, the primary and secondary peaks do not always align perfectly. This can result in secondary peaks that are more than the threshold not being called as mixed if the secondary peak location is offset from the primary. Second, the relative processed peak heights do not always correspond to the relative signal strength seen in the raw data channel. This is an issue for both KB and PeakTrace 5, but it is a larger issue with PeakTrace 5.

To overcome both of these problems the algorithm used for mixed basecalling in PeakTrace 6 was improved to ensure that the secondary and primary peaks are aligned before making the relative signal height comparisons. The new algorithm also uses the relative signal heights from the raw data channel to make the peak height comparisons.

Figure 2. Polymorphic region basecalled using PeakTrace 5 and a 30% threshold.

Figure 2. Polymorphic region basecalled using PeakTrace 5 and a 30% threshold.

Figure 3. Polymorphic region basecalled using PeakTrace 6 and a 30% threshold.

Figure 3. Polymorphic region basecalled using PeakTrace 6 and a 30% threshold.

The effect of these changes can be seen in Figures 2 & 3. The mixed bases at positions 196 and 197 were not detected as mixed basecalls by PeakTrace 5 due to the peak offsets and secondary peak height suppression in the processed data channel. These problems do not occur with the new mixed basecall algorithm introduced in PeakTrace 6 and both positions are now called as mixed.

This problem of secondary peak height suppression is very obvious when the no peak resolution PeakTrace output is used (Figure 4). This setting outputs the raw data smoothed, baselined and mobility shifted. The peaks while not as well resolved in the late regions of the trace show the raw data relative peak heights.

Figure 4. Polymorphic region basecalled using no peak resolution and a 30% threshold.

Figure 4. Polymorphic region basecalled using no peak resolution and a 30% threshold.

If desired the old PeakTrace 5 mixed basecalling module can be used by checking simple mixed basecalling in the Additional Options window along with using the appropriate mixed peak threshold. This will make PeakTrace 6 perform the same mixed basecalling as was done in PeakTrace 5.

Potential Pitfalls with Mixed Basecalling

The major factor to be aware of when using the new PeakTrace 6 mixed basecall setting is the best mixed peak threshold to use will likely have changed. If you had previously been using a mixed peak threshold value of 50% then you may wish to reduce the value to 30%. It is hard to give a precise mixed peak threshold level to use for mixed basecalling, but setting the mixed peak threshold high (over 50%) will result in fewer false positives and the expense of more false negatives and vice versa. A good starting point for the mixed peak threshold is 30%, but you may wish to change this threshold on the basis of your needs.