Nucleics

Nucleics

Auto PeakTrace RP 6.46 Released

By

Auto PeakTrace 6 RP

Auto PeakTrace 6 RP

We have released Auto PeakTrace RP 6.46. This is an bug fix and enhancement release focused on improving the processing of mixed or polymorphic traces. It is a recommended upgrade for all PeakTrace RP users.

The complete list of changes in this release:

  • Bug fixes and enhancements for the clean baseline module to avoid the removal of true mixed peaks.
  • Improved processing of short failed or mixed traces.
  • Improvements to mixed basecalling to improve detection of true mixed peaks.
  • Improvements to fuse basecall option. Can now be set up to base 999.
  • Improvements of the processing of PCR product traces.
  • New error code: 59. Mixed trace.

Auto PeakTrace RP 6.46 for Windows and MacOS X can be downloaded from the PeakTrace RP downloads page. This release will also be available via the automatic update function in the next few days.

If you require the password to access the RP 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.

In Depth: Use Edited Basecalls

By

One of the new features introduced in PeakTrace 6.10 was the use edited basecalls setting. This option allows the retention of manual basecalling changes to the sequence when the fuse trace or fuse basecalls settings are used. This setting can be useful if you wish to correct KB Basecalling errors before PeakTrace Basecalling and the result is best illustrated by example.

Figure 1 shows a KB basecalled trace file. Note the basecalling error made by KB at base 6 where an obvious G peak was called as a low quality T.

Figure 1. KB basecalled trace before manual editing.

Figure 1. KB basecalled trace before manual editing.

Figure 2 shows the same trace after manual editing using Chromas. Note the T at base 6 has been manually changed to a g.

Figure 2. KB basecalled trace after manual editing to correct basecalling errors.

Figure 2. KB basecalled trace after manual editing to correct basecalling errors.

Figure 3 shows the manually edited trace after PeakTrace Basecalling using the default fuse trace setting of 1, but without the use edited basecalls setting. Note the edited G base at base 6 has reverted to the original KB basecalled T.

Figure 3. PeakTrace Basecalled trace using fuse trace of 1 without the use of use edited basecalls.

Figure 3. PeakTrace basecalled trace using fuse trace without use edited basecalls.

Figure 4 shows the manually edited trace after PeakTrace Basecalling using the default fuse trace setting of 1, but with the use edited basecalls setting. Note the edited G base at base 6 has been retained in the final call.

Figure 4. PeakTrace basecalled trace using fuse trace and use edited basecalls.

Figure 4. PeakTrace basecalled trace using fuse trace and use edited basecalls.

It is important to understand that the use edited basecalls only applies to the KB basecalled sequence retained by the fuse trace or fuse basecall options. Any manually edited basecalls that are subsequently replaced with PeakTrace basecalls are not kept.

Of course any PeakTrace basecalled trace can be manually edited after basecalling just like KB. If you wish to retain more of the manually edited basecalls then you can use a larger value for the fuse trace or fuse basecall setting (e.g. use 300 instead of 1).

Auto PeakTrace RP 6.43 released

By

Auto PeakTrace 6 RP

Auto PeakTrace 6 RP

We have updated Auto PeakTrace RP to version 6.43. Most of the changes for this release are in the backend server so everyone using PeakTrace RP will get the improvements automatically, but there is a local client fix for a warning bug when auto start is used.

The full list if changes are

  • Bug fixes for the auto start and clean baseline modules.
  • Improvements to fuse basecall module.

Auto PeakTrace RP 6.43 for Windows and MacOS X can be downloaded from the PeakTrace RP downloads page and it will also shortly be available via the automatic update function. If you require the password to access the RP downloads page please contact us @ {This email is obscured. Your must have javascript enabled to see it} or get in contact your local supplier for the update.

In Depth: Secondary Noise Peaks

By

One of the new features introduced in PeakTrace 6.40 was secondary noise peaks. The setting is unusual in that it actually make the traces appear to be of lower quality than the result normally provided by PeakTrace. This setting introduces artificial secondary peaks under the primary peak proportional in height to the percentage value entered and the height of the primary peak. For example, using a secondary noise peaks value of 5 adds secondary peaks that are 5% of the primary peak heights under every primary peak in the trace.

Adding secondary noise peaks can be useful if you wish to give your end users a trace file that looks much closer to that which would be output by the KB Basecaller. Figures 1 to 3 show examples of how the secondary noise peaks option works. Figure 1 shows an original KB Basecalled trace at ~base 400; Figure 2 shows the same trace after PeakTrace basecalling with a secondary noise peaks setting of 0 (i.e. off); Figure 3 shows the same trace, but using a secondary noise peaks setting of 5 (i.e. 5%).

kb_400

Figure 1. KB basecalled trace.

pt_nsp_0_400

Figure 2. PeakTrace basecalled trace with default processing values.

pt_nsp_5_400

Figure 3. PeakTrace basecalled trace with a secondary noise peaks setting of 5.

 

While it is immediately obvious that the standard PeakTrace basecalling (Figure 2) is far superior to that provided by KB (Figure 1), some users are concerned that the default PeakTrace basecalled traces look “too good” to be real. While we think this is an unnecessary concern (after all nobody complains that their dinner tastes “too good” and they wished that it tasted worse), we do understand that some of our customers want to provide their end users with the superior basecalling of PeakTrace with a trace that looks very similar to what KB generates. The secondary noise peaks setting provides a simple way of degrading the trace output so that PeakTrace basecalled traces look like KB basecalled traces, while still providing the same base and quality score improvements that PeakTrace offers.

It is advisable to not use a secondary noise peaks value over 10. Using a large value will create a trace with a very poor appearance and end users may mistake the secondary peaks as true mixed peaks. This is one setting to use with caution.

 

In Depth: Set ABI Limits

By

One of the new features introduced in PeakTrace 6.40 was Set ABI Limits. This option changes the PeakTrace trimming functions from hard trims (i.e. actual removal of the low quality regions) to the same soft (virtual) trimming that is performed by the KB Basecaller’s Post-Processor module. If trace trimming is selected within Sequencing Analysis, then KB will remove the low quality regions from the trace by setting the ABI Limits values in the trace file to indicate the high quality region. When the trace or basecall is viewed in Sequencing Analysis or Sequence Scanner, the trimmed basecalls are still present, but the peaks and bases are now grey (Figure 1).

Sequencer Scanner with no ABI limits range set.

Figure 1. Example trace viewed in Sequence Scanner with the ABI limits range set. The ABI limits region starts at base 15.

PeakTrace is now also able to set the ABI Limits values by using the Advanced Option Set ABI Limits with trace trimming. Instead of the hard removal of the low quality regions, these regions are “greyed out” when viewed in Sequence Scanner or Sequencing Analysis – just like KB (Figures 2 & 3).

abi_limit_sequence_scanner_far_end

Figure 2. PeakTrace basecalled trace using Set ABI Limits and Q Average Trim of 12 & 20. The last low quality region has been greyed out.

abi_limit_sequence

Figure 3. PeakTrace basecall using Set ABI Limits and Q Average Trim of 12 & 20. The last low quality bases are shown in grey and the high quality regions in blue.

One of the limitations with using Set ABI Limits is that none of the non-ABI trace viewers are able to use the ABI limits region. For example, Figure 4 shows the same trace as shown in Figure 2, but in FinchTV. The low quality region after base 1390 is not shown greyed out or trimmed by FinchTV.

abi_limit_finch_tv_end

Set ABI Limits is a very useful addition for those users of PeakTrace who wish to replicate the results provided by the KB Basecaller as close as possible. It provides the same “greyed out” trimming of the low quality regions that Sequencing Analysis Post-Processing provides.

Users of this setting are encouraged to upgrade to PeakTrace 6.42 or greater as this release has introduced a workaround for a bug in Sequencing Analysis that stops the display of the 3’ trimmed region when the 5’ end is untrimmed.

Auto PeakTrace RP 6.42 Released

By

extra baseline processing

We have released Auto PeakTrace RP 6.42. This release is an optional minor release that improves the basecalling of traces with large spikes as well as the use abi limit option. The full list of changes in this release are

  • Improved processing of traces with large peaks.
  • Change to the abi limits module to make the minimum start base 1 rather than 0. This fixes a display bug for traces within ABI’s Sequence Analysis™ software.
  • Updated list of supported sequencing run conditions.

Auto PeakTrace RP 6.42 can be downloaded from our PeakTrace RP Downloads page for both Mac OS X (>10.8) and Windows (XP, Windows 7, Windows 8 and Windows 10). It will bye available via the PeakTrace RP automatic application update function shortly. 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 to create a PeakTrace account.

Auto PeakTrace RP 6.41 Released

By

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 RP 6.40 Released

By

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.

Auto PeakTrace RP 6.30 Released

By

apt_rp_630_options_window

We have released Auto PeakTrace RP 6.30. This is a major update with two new features – extra baseline and secondary noise peaks. These two setting can improve the appearance of the traces and help to better match the appearance of PeakTrace basecalled traces to that obtained from the KB Basecaller. These new features are explained in the user help manual, but we will also be writing in depth articles on each of these new features. The full list of changes include.

  • New Feature. Extra baseline option for improved appearance of trace files.
  • New Feature. Secondary noise peak option to add peak noise to the traces.
  • Unsupported run types now output .seq and .phd.1 files based on the KB sequence if these file types are requested.
  • The logfile now records the local time rather than the server time of processing.
  • Further improvements to increase the robustness of the software on very fast networks.
  • 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 sequence will be basecalled.
  • Upgrade of 7za to 15.14.
  • Bug fixes and other enhancements.

This PeakTrace RP release can be downloaded as per usual from the PeakTrace RP Downloads page for both MacOS X (10.6+) and Windows (XP, Windows 7, Windows 8 and Windows 10). It will be also be available via the in-app update function in a few days. 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 user of Auto PeakTrace RP 6 then please visit the Auto PeakTrace 6 RP registration page.

Auto PeakTrace RP 6.24 Released

By

nucleics_10

We have released Auto PeakTrace RP 6.24. This update improves the robustness of Auto PeakTrace RP especially for user with fast networks. It is a recommended  that all user to upgrade to this release. The full list of changes are

  • Improvements to increase the robustness of the software on very fast networks.
  • Improved cleaning of temporary files from the Application Data folder.
  • Upgrade of cURL to 7.48.0.
  • Other minor bug fixes and enhancements.

This release 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+) and automatically 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 register on the Auto PeakTrace 6 RP registration page.