Nucleics

Nucleics

PeakTrace 6.92 Released

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KB PeakTrace Comparison

We have released PeakTrace 6.92. This is a bug fix and enhancement release and is the culmination of six months hard work by the team at Nucleics. We have gone through nearly every line of code within PeakTrace to improve and enhance the processing and basecalling of Sanger sequencing traces. While there is no single massive changes from all this work, the cumulative effect of all these small changes adds up to a major improvement. The full list of changes includes.

  • Support for additional run conditions including ultra low voltage runs.
  • ABI 377 support.
  • Improved basecalling.
  • Improved basecalling and processing of PCR product traces.
  • Improved mixed basecalling.
  • Improved processing of traces with polymorphic peaks.
  • Improved processing of traces with long single base runs.
  • Improved trace trimming.
  • Improved removal of air bubble spikes in the trace.
  • Improved clean baseline and extra baseline processing.

PeakTrace 6.92 is live on our servers so our PeakTrace RP users will be automatically getting the benefits of 6.92. Over the coming week we will be releasing updates for the standalone PeakTrace:Box for Linux and Windows, along with an update for the Auto PeakTrace RP client program for Windows and MacOSX. We will make further announcements when these updates are released.

In Depth: Input Overwrite

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A new feature introduced in Auto PeakTrace 6™ is input overwrite.

Under normal circumstances, attempting to set the input and output folder will result in an error when you process the traces.The aim is to prevent the accidental overwriting of the original input .ab1 files with the PeakTrace basecalled version. If you wish to overwrite the input (i.e. have the input and output folders to be identical) you need to select input overwrite. This option is off by default.

input-error

Figure 1. When input overwrite is off any attempt to process traces when the input and output folder are set to the same directory will produce an error.

 

Figure 2. When input overwrite is on the input trace files can be overwritten by the PeakTrace processed traces.

Potential Pitfalls

  • You need to be very careful when using this feature as the original KB basecalled trace file will be overwritten. If you don’t want to lose your original data then you should leave this option off, or retain a copy of the original .ab1 data elsewhere. While the original trace file can be regenerated by reprocessing through either the KB or ABI Basecallers, if you also use noise raw data there will be no way to regenerate the original trace file.
  • It would be a good idea to archive your traces when using input overwrite to avoid the potential for wasting units on reprocessing data.

In Depth: Force Processing

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One of the more commonly misunderstood setting within PeakTrace™ is the force processing option. This option will allow you to force PeakTrace basecalling of files that would normally be rejected by PeakTrace for poor quality under normal circumstances (Figures 1 & 2).

Figure 1. An example low quality trace that PeakTrace fails under default conditions.

 

Figure 2: Successful PeakTrace basecalling of the same trace after force processing has been toggled on.

Advantages

  • Force processing may be useful when you wish to proceed with processing poor quality traces that are not able to basecalled by PeakTrace under standard basecalling settings.
  • Forced processing may allow PeakTrace to rescue some usable data from an otherwise unusable trace (Figures 2 & 3).

 

Figure 3. A side by side comparison of the original trace, and the force processed trace. The original trace failed to be basecalled by KB, and was also rejected by PeakTrace under the default settings due to poor quality. Force processing has allowed the PeakTrace basecalling and trace processing to proceed.

 

Figure 4: Blast alignment of the extracted data reveals a sequence match. Force processing has allowed useful data to be obtained.

Potential Pitfalls

While force processing can be useful to rescue some traces, there are risks with using it on all traces.

  • Force processing may produce nonsensical or corrupted trace files. It is advised that you use this feature only as a last resort, and that you do so with additional caution.
  • Force processing is not magic. It can not create a high quality trace from a failed reaction. In most cases, a poor quality trace indicates that the sample will need to be sequenced again under improved sequencing conditions.
  • Force processing may result in PeakTrace crashing. While PeakTrace can automatically recover from a crash and restart, you may notice the program closing and restarting.

It is advised that force processing is only used if needed and not by default on all traces.

Support for purchasing PeakTrace in local currencies

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We have recently added support for buying PeakTrace RP units in your local currency with local VAT/GST/Sales taxes include via paddle.com. The list of currencies that can be used includes: Euro (EUR), British Pound (GBP), Chinese Yuan (CNY), Polish Zloty (PLN), Swedish Krona (SEK), South African Rand (ZAR), Swiss Franc (CHF), Brazilian Real (BRL), Ruble (RUB), Australian Dollar (AUD), Canadian Dollar (CAD), Hong Kong Dollar (HKD), Singapore Dollar (SGD), New Zealand Dollar (NZD), Danish Krone (DKK), Hungarian Furint (HUF), Czech Koruna (CZK), Indian Rupee (INR).

While this new way of buying PeakTrace RP units can be advantageous in some situations, the downside is the cost is greater due to the extra fees charged by Paddle. The major use case is for customers located in India who can now use a local Indian credit card to buy PeakTrace RP units without the need for an international credit card. For most other customers the current US$ payment system (handled by Paypal) is likely be best option.

Auto PeakTrace RP 6.91 Released

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Auto PeakTrace 6 RP 6.91

Auto PeakTrace 6 RP 6.91

We have released Auto PeakTrace 6 RP 6.91 for Windows and MacOS X. This update is an “under-the-hood” update move our primary development platform from Windows XP to Windows 10. Auto PeakTrace RP still supports Windows XP, but by updating we aim to improve the performance and compatibility of PeakTrace with newer version of the Windows OS. The full list of changes include.

  • New Feature. x64 version.
  • New Feature. High DPI monitor support.
  • New PeakTrace installer.
  • Further improvements to the processing and basecalling of weak traces.
  • 7za updated to 18.05.
  • cURL updated to version 7.59.0.
  • Wine updated to 2.2 for the MacOSX release.
  • Other minor bug fixes and enhancements.

Auto PeakTrace RP 6.91 can be downloaded from the PeakTrace RP Downloads page. If you do not know the password to access the PeakTrace RP Downloads page please contact us or your local supplier for the update.

Auto PeakTrace RP 6.90 Released

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Auto PeakTrace RP 6.90 Options

Auto PeakTrace RP 6.90 Options

We have released Auto PeakTrace RP 6.90. This is a very large upgrade and a significantly improved version of the PeakTrace Basecaller. We have spent the last 6 months optimising all the algorithms and code focused on improving the read length and appearance of poor quality traces. We will be writing about the many improved features over the coming weeks.

The full list of changes includes.

  • New Feature. Support for the ThermoFisher SeqStudio™ Sequencer.
  • New Feature. Fuse trace at base default now 0.
  • Improved basecalling especially of low quality traces.
  • Improved trace processing.
  • Improved peak normalization.
  • Improved clean baseline.
  • Improved detection and basecalling of PCR product traces.
  • Improved PeakTrace processing and basecalling through “dye blobs” peaks.
  • The signal start peak auto now sets the start peak to the value closest to the model expected value.
  • The automatic fuse trace at base or fuse basecalling at base on traces with large dye blobs peaks can be turned off by setting fuse basecall to -1.
  • An additional secondary PeakTrace RP server established in China.
  • Unit data and build date can be written to a text file with the -rf options.
  • Support temporary removed for the following machines and run types. These run conditions are rarely used, but support will be returned in the next major update.
    • ABI 3700 runs
    • MegaBACE 1000 & 4000 runs
    • ABI 3130 80cm array runs
    • ABI 3730 runs below 6kV
    • ABI 310 36cm & 50cm array runs
    • ABI 310 30cm array runs with a voltage below 12 kV
    • All ET terminator runs
    • MJ Research Biostation runs
  • Bug fix that prevented clear range trim from being applied to the front of the trace.
  • Many other bug fixes and internal improvements.

Auto PeakTrace RP 6.90 can be downloaded from the PeakTrace RP Downloads page. If you do not know the password to access the PeakTrace RP Downloads page please contact us or your local supplier for the update. User of older versions should be notified by the automatic update feature.

This update is highly recommended for all users of the Auto PeakTrace RP or earlier.

An update for the standalone box versions of PeakTrace will be released in the next few days.

Auto PeakTrace RP 6.82 Released

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Auto PeakTrace RP 6.82 Options Window

Auto PeakTrace RP 6.82 Options Window

We have released an update for Auto PeakTrace RP (6.82). This is an intermediate release, mostly bug fixes and improvements, but it also has a couple of new features that are of interest for some of our customers. The full list of changes and improvements include:

  • New feature. Ultra-aggressive clean baseline cleaning when mixed peak threshold is set to 102.
  • New feature. Support added for:
    • ABI3730 ultra-rapid 50cm arrays runs
    • ABI3730 10.5kV50cm array runs
  • New Feature. European located secondary server ensuring data does not leave the EU.
  • New feature. Output both peaktrace processed and no peak resolution traces at the same time.
  • Improvement to basecalling include G-peak dropouts.
  • Bug fix that prevented the input and output folders being updated from the Auto PeakTrace RP 6 command line.
  • Improved basecalling and detection of low quality mixed peak traces.
  • Improved trace normalization.
  • Bug fix for trace trimming modules. On traces that are not improved, only soft trimming (abi limits) is applied. If the abi/kb basecaller is selected and set abi limit is unchecked then the trace will still be hard trimmed.
  • Version, build date are accessible from the Auto PeakTrace RP 6 command line.
  • Errors and processing progress when using Auto PeakTrace RP 6 from the command line are now output to the command line window.
  • Other bug fixes.

Auto PeakTrace RP 6.82 for Windows and (MacOS X will be released later) and can be downloaded from the PeakTrace RP downloads page. This release is also be available soon via the automatic update feature within Auto PeakTrace RP if you are using a recent version of Auto PeakTrace RP.

If you require the password to access the PeakTrace RP downloads page please contact us @{This email is obscured. Your must have javascript enabled to see it} or your local supplier.

We will be releasing an update for the PeakTrace:Box version in the next few days.

Auto PeakTrace & PeakTrace 6.81 Released

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We have released a minor update for Auto PeakTrace and PeakTrace (6.81). The full list of changes and improvements include:

  • New Feature. Support added for
    • ABI 3730 ultra rapid 50cm arrays runs
    • ABI 3730 10.5kV 50cm array runs
  • Improved basecalling and detection of low quality mixed peak traces.
  • Improved trace normalization.
  • Bug fix for trace trimming modules.
  • With traces that are not improved, only soft trimming (abi limits) is applied. If the abi/kb basecaller is selected and set abi limits is unchecked then then the trace will still be hard trimmed.
  • Other bug fixes.

Auto PeakTrace 6.81 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 for the update. The command line PeakTrace 6.81 for Windows and Linux is also available on the update pages.

This update is an optional update for all users of the Auto PeakTrace or PeakTrace 6.80. For user of Auto PeakTrace the changes are automatically applied on the server end.

Auto PeakTrace RP 6.80 Released

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Auto PeakTrace RP 6.80 Options Window

Auto PeakTrace RP 6.80 Options Window

We have released an major feature release for Auto PeakTrace RP (6.80). The full list of changes and improvements include:

  • New Feature. Total traces processed and success rate shown.
  • New Feature. Auto PeakTrace RP 6 can be used as a command line program.
  • Improvements to fuse basecall and fuse trace with difficult sequences.
  • Improvement of the basecalling of “drop out” peaks.
  • Improvements to extra normalization of PCR product traces.
  • More accurate estimated time for trace processing completion.
  • Other bug fixes and enhancements.

The major change of this release is that Auto PeakTrace RP 6.80 can now be used from the command line. This allows the easy integration of Auto PeakTrace RP 6.80 into various LIMS scripts.

If Auto PeakTrace RP 6.80 is used from the command line with no options it will use the settings (including the input and output directories) saved from the last use of Auto PeakTrace RP 6. The saved settings can be overwritten by the use of command line switches. If no switch value is provided on the command line then value in the save setting file will be used. Settings that are changed via the command line update the setting file for future runs of Auto PeakTrace RP 6.

It is recommend that if you wish to use Auto PeakTrace RP 6 in a command line script that you first contact Nucleics @ {This email is obscured. Your must have javascript enabled to see it} or your local supplier for advice.

Auto PeakTrace RP 6.70 for Windows and (MacOS X will be released later) and can be downloaded from the PeakTrace RP downloads page. This release is also available via the automatic update feature within Auto PeakTrace RP if you are currently using a recent version.

If you require the password to access the PeakTrace RP downloads page please contact us @{This email is obscured. Your must have javascript enabled to see it} or your local supplier.

Auto PeakTrace 6.71 & PeakTrace 6.71 Released

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Auto PeakTrace 6.71 Options Window

Auto PeakTrace 6.71 Options Window

We have released an improvement/bugfix release for Auto PeakTrace and PeakTrace (6.71). The full list of changes and improvements include:

  •  Improved processing of weak traces.
  • Improvement to q count trim and n base trim modules.
  • Fix of a bug that resulted in some good peaks being called as N.

Auto PeakTrace 6.71 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 for the update. The command line PeakTrace 6.71 for Windows and Linux is also available on the update pages.

This update is a recommended update for all users of the PeakTrace:Box System. For user of Auto PeakTrace RP all changes are on the server side and so you have been automatically updated.