Nucleics
Nucleics will be exhibiting at the Association of Biomolecular Resource Facilities 2017 Annual Conference in San Diego from March 25th to 28th. Please drop by and see the Nucleics team at Booth #125 as we would love to chat to you about your research and introduce you to some of our exciting new products we will be releasing this year.
We have released a small update for Auto PeakTrace (6.52) to fix a bug in the copy other files option introduced in Auto PeakTrace 6.47 that prevented the non .ab1 files from being copied to the output folder when located in sub-folders. This bug only affects Auto PeakTrace on Windows and is optional if you are not using the copy other files setting.
This release also updates the required CodeMeter Runtime to 6.40b. This release from Wibu fixes a bug specific for Windows XP users that may cause the license to no longer work.
It is possible that the PeakTrace Serial Number will need to be entered again after installation. If so, just press the back button on the registration window and enter your existing license serial number.
Auto PeakTrace 6.52 can be downloaded as usual 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.
We have released a small optional update to Auto PeakTrace (6.51) as a work around to a bug recently introduced in the CodeMeter runtime. This bug only affects Auto PeakTrace on Windows.
If you have already upgraded to Auto PeakTrace 6.50 then you only need to install this update if we advise you to do so. For anyone using a version of Auto PeakTrace before 6.50 we strongly encourage you to update to this release.
Auto PeakTrace 6.51 can be downloaded as usual 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.
Auto PeakTrace 6.50 Options Window
PeakTrace 6.50 for Linux, PeakTrace 6.50 for Windows, and Auto PeakTrace 6.50 have been released. This update improves the performance and accuracy of PeakTrace and incorporates improvements to the QualTrace III Trace Reports to make them independent of PeakTrace. This release is a recommended update for all users of PeakTrace. The full list of changes
PeakTrace 6.50 for Linux, PeakTrace 6.50 for Windows, and Auto PeakTrace 6.50 can be downloaded as usual from the PeakTrace:Box Downloads page. The automatic update for Auto PeakTrace will be released in the coming days.
The two command line versions (PeakTrace 6.50 for Windows or Linux) require a password to unzip the files so please contact us @ {This email is obscured. Your must have javascript enabled to see it} or your local supplier for the archive password (this is different to the password to access the downloads page). You will need to download and use the free program 7zip to unzip the archives.
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. This update will also be available via the automatic update system soon if you are using PeakTrace 6.20 or higher.
If you are currently running a version of PeakTrace from before 6.10 you will also need a license upgrade and/or the PeakTrace Serial Number. Please contact us to obtain these updates.
The changes and improvements of this release have also been provided to the RP servers so users of Auto PeakTrace RP will obtain them automatically.
The q average trim option allows you to select a quality score average clipping threshold and window size, so that traces are clipped at their 5′ and 3′ ends when the average quality score in a sliding window of defined base number falls below the clipping threshold. The default values are a quality score average clipping threshold of 12, and a sliding window of 20 bases. This means that by default, all traces are clipped when the average quality score falls below 12 within a window of 20 bases.
This option is especially useful for removing low quality regions at the 5′ and 3′ ends of your traces. Figure 1 shows an unclipped trace with a poor quality 3′ region and Figure 2 shows the same trace processed using the q average trim to remove the low quality region.
Figure 1. An unclipped trace showing a poor quality 3′ region.
Figure 2. The same trace as in Figure 1, but trimmed with the Q average trim function. Selected parameter values were an average clipping threshold of 15, a sliding window of 20 and the trim 3′ end only function toggled on.
The q average trimming option is compatible with the set ABI limits option, which will perform a soft or “virtual” trim of low quality end regions.
If you have untrimmed traces that have already been processed through PeakTrace and you would like to trim them without being charged more units, you can perform post-process trimming by selecting abi/kb as the basecaller and choosing the appropriate trimming method. Remember to turn off the clean baseline, extra baseline and extra normalization options or else you will be charged for trimming the traces.
Auto PeakTrace™ 6 (Windows version only) offers support for processing files located inside folders containing unicode characters (non-English), plus the ability to putput unicode logfiles. This allows the logfile to be output in Unicode, rather than ASCII text. Under normal circumstances, any Unicode characters will be rendered as a “?” in the logfile (Figure 1), but with this option selected, the logfile will display the Unicode path characters (Figure 2).
Figure 1: The log file of a run where the selected input and output folder were in unicode, but unicode logfile was toggled off.
Figure 2: The same file as in Figure 1, but output with unicode logfile toggled on.
Considerations
The skip short traces allows PeakTrace 6™ to skip over traces that have less than a certain number of bases and which may not be improved by PeakTrace. This setting is useful because the KB Basecaller typically basecalls short read DNA sequence well and hence the improvement from using PeakTrace can be minimal. By using this setting users can avoid having to manually preselect the traces for PeakTrace basecalling, a time consuming and often inaccurate step.
This function is aimed at users who are only interested in improving longer runs, or would like to avoid having to manually preselect the traces that will see the most improvement from PeakTrace. The reads below the set read length are skipped over and the cost of using PeakTrace avoided on these traces.
When using this function, trim settings, noise raw data and stealth mode will all still be applied to the short trace at no charge (Figures 1 & 2). If it is desired that trimming is not performed on the short traces then the trim improved traces only function should be selected. Trim improved traces only will apply the trimming to the PeakTrace processed traces only and not to the short traces skipped over.
Figure 1. KB basecalled short trace before processing
Figure 2. Trace skipped by Auto PeakTrace 6. Note that this trace has had trimming applied, however the rest of the trace is identical to the original file
If the skip short/pcr base option is set above 500 bases then traces that could have been improved will be charged. A setting lower than 500 will allow more traces to be processed and the functionality of the skip short traces option will remain on, however a setting of 0 in the skip short/pcr base option will effectively turn skip short traces off, as processing will be attempted for all traces with more than 0 bases.
If this setting is used with those settings that improve the KB traces (i.e. clean baseline, extra normalization, and extra baseline)then PeakTrace will improve the KB trace and this will become a charged trace. To avoid this, set the basecaller to peaktrace and/or unselect any of KB improving settings.
Auto PeakTrace 6.47, PeakTrace 6.47 for Windows, and PeakTrace 6.47 for Linux x86_64 have been released. This update is a bug fix and optimisation release that resolves a number of outstanding bug and small annoyances. It is a recommended update for all users of PeakTrace 6.40 or greater.
Auto PeakTrace 6.47, PeakTrace 6.47 for Windows, and PeakTrace for 6.47 for Linux x86_64 can be downloaded from the PeakTrace:Box Downloads page.
The two command line versions (PeakTrace 6.47 for Windows or Linux) require a password to unzip the files so please contact us @ {This email is obscured. Your must have javascript enabled to see it} or your local supplier for the archive password (this is different to the password to access the downloads page). You will need to download and use the free program 7zip to unzip the archives.
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. This update will also be available via the automatic update system soon if you are using PeakTrace 6.20 or higher.
If you are currently running a version of PeakTrace from before 6.10 you may also need a license upgrade and/or the PeakTrace Serial Number.
One of the more commonly misunderstood settings within PeakTrace™ is true profile. By default PeakTrace uses a flat profile which normalizes the processed peaks to approximately the same height irrespective of the true signal intensity in the raw data channels (Figure 1).
Figure 1. Example trace following basecalling and processing with PeakTrace under default settings.
The true profile setting of PeakTrace works in exactly the same way as the True Profile setting within ABI’s Sequence Analysis software. Selecting true profile outputs traces where the processed peak heights reflect the underlying peak intensity in the raw trace data channels. When traces are processed using true profile, samples that show a large peak signal decline will have large peaks at their 5′ ends which tapers off to small peaks or baseline noise at the 3′ end (Figure 2). The use of true profile will ensure that the processed peak signal reflect the signal strength profile seen in the raw trace data (Figure 3).
Figure 2. The same example trace as shown in Figure 1. with true profile selected
Figure 3. The raw data from the example trace shown in Figures 1 and 2.
The main advantage of using true profile is it allows users to quickly visualize any problems stemming from rapid peak signal decline. This can prove useful in helping identify problems that have occurred in sample preparation which the flat profile may hide. The default for true profile is off.
The major pitfall with using true profile is the peaks at the 3′ end of the trace can be very small if the trace signal declines very rapidly (i.e. a so called “ski-slope” trace).
It is important to note that true profile will not be set if extra normalization is also chosen. Be sure to uncheck extra normalization if you want to output the true profile.
A question we occasionally get asked is how to prevent users from accidentally reprocessing traces if they open them in Sequence Analysis. Since PeakTrace does not change the raw data in the .ab1 file by default, it is possible to reprocess the trace files through the KB™ or the ABI Basecaller again. This will regenerate the original pre-PeakTrace trace, thus losing the PeakTrace sequence. This problem can be avoided by using the Noise Raw Data option.
The Noise Raw Data setting replaces the raw data channels with a random signal that prevents the accidental reprocessing of traces by KB or the ABI Basecaller.
Figure 1 shows an example of the normal raw data channels contained within a .ab1 trace file. Figure 2 shows the raw data of the same trace after using the Noise Raw Data setting. Attempting to re-basecall the .ab1 file using KB will result in a KB processing error, thus preventing the PeakTrace basecalls and peaks from being lost.
Figure 1. Example of raw data channels contained within a .ab1 trace file.
Figure 2. Example of the raw data channel of use of Noise Raw Data setting.
It is strongly recommended that if this setting is used that the the original .ab1 files are kept, since in the event that the KB version of the trace file is is required, there is no way to recover it once this option has been applied. This setting is off by default.
