Introduction
PeakTrace™ is a DNA sequencing basecaller offering improved read lengths for DNA sequencing traces collected on the Applied Biosystems ABI 377, 310, 3700, 3730, 3730xl, 3100 and 3130 and MegaBACE DNA sequencers. The PeakTrace basecalling software offers the following features:
- Up to 100% more high quality bases per trace.
- Compatibility with all ABI DNA sequencers.
- A simple, easy to use, FTP based user interface.
- A "success only" fee. This means that you will only pay for traces that have at least an extra five high quality bases.
- PeakTrace is also offered as a standalone version (PeakTrace:Box) for those users who wish to use the basecaller at their own facility.
PeakTrace Basecalling Software
Current state-of-the-art automated DNA sequencers, like the ABI 3730 series, can collected sequencing reads that easily exceed 1100 nucleotides. Unfortunately, such sequences suffer from broad, overlapping peaks and low resolution in the last part of the trace. This latter sequence region is associated with low base quality scores and high base call error rates.
The PeakTrace basecalling software uses a new approach to extract high quality sequence data from the poorly resolved regions of each trace. The PeakTrace software extracts and reprocesses the raw data contained within each ABI trace file using a series of highly sophisticated algorithms developed by Nucleics. This data reprocessing enables the basecaller contained with the PeakTrace software to make significantly better base and quality calls on the trace. An example of PeakTrace basecalling is shown in Figure 1.
Figure 1. Comparison of the PeakTrace basecaller with the KB basecaller. This example is from an ABI 3130 trace and shows the region from base 1130 to 1180.
PeakTrace basecalling can be performed any time after the trace data has been collected. This allows it to be used with previously obtained data sets such as partial genome sequencing projects data. By applying PeakTrace to such trace data and reassembling the sequences, the final genome assembly can be significantly improved without the need to collect more sequence data. Additionally, as the output of the PeakTrace service is a standard scf trace file, it means that PeakTrace can be used with standard downstream software application such as Staden or Phrap.
Features of the PeakTrace basecalling software
Easy to use
The PeakTrace FTP basecalling service is simple to use. After opening an account, users log in to our ftp server using any standard ftp program. Users select the ABI trace files for PeakTrace basecalling and upload them to the PeakTrace folder contained within their account on our ftp server. Files can be uploaded as individual files, zip archives, or as complete folders of traces files. Once uploaded, the PeakTrace software automatically basecalls the trace files. When complete, an email is sent to users letting them know that the trace data set has been basecalled. user then log into their ftp accounts and download the improved traces (these are located in the "out" folder).
The PeakTrace:Box basecalling system is equally easily to use. The system is based around a small, silent computer (an Apple Mac Mini running Windows XP Professional). The PeakTrace system comes with an easy to use graphical software (Auto PeakTrace) to allow the automatic processing of the traces. In addition, the Auto PeakTrace software provides trace improvement reports for both the individual user and the facility administrator making it straightforward to integrate into a core lab environment where users are charged on a per trace basis.
The PeakTrace basecaller is compatible with trace files collected from all ABI DNA sequencing instruments including the ABI 3700, ABI 310, ABI 377, ABI 3100, ABI 3130, ABI 3730 as well as the MegaBACE. PeakTrace is compatible with rapid run traces collected at high run voltages (>12kV).
The PeakTrace basecaller can be tested using the free PeakTrace basecalling service. This service provides the exactly same basecalling as the paid ftp based service, however, users are limited to processing only 10 traces per day. If you have a large number of traces to process then we recommend that you open an account to use the high throughput PeakTrace service.
Increases trace read length and reduces errors
The PeakTrace Basecaller provides the user with significantly longer and higher quality reads (Figures 1 & 2). These improved read lengths mean that fewer sequencing reactions need be performed
to complete a sequencing project, thus saving the investigator both time and money.
Figure 2. Comparison of the PeakTrace basecaller with the standard ABI basecaller from a ABI 377 trace file.
The improved DNA sequencing read lengths provided by the PeakTrace basecaller increase sequencing throughput and significant lower the overall per base cost. In addition, these longer read lengths enable sequencing projects (e.g. whole-genome, cDNA clones, etc) to be completed at lower sequence coverage levels, thus offering further cost- and time-savings.
For further information on PeakTrace please or visit our PeakTrace forum.