Introduction
LongTrace™ is a DNA sequencing system for increasing the read length of DNA sequencing traces collected on the Applied Biosystems 3730, 3730xl, 3100 and 3130 DNA sequencers. The LongTrace sequencing service offers the following features:
- Up to 30% more KB base called Q20+ bases per trace, with an average improvements of 12% to 20%.
- Complete compatibility with the KB base caller for traces collected on the ABI 3730, 3130 and 3100 series of DNA sequencing instruments.
- A simple, easy to use ftp based user interface making it easy to upload large numbers of files.
- A "success only" fee. This means that you will only pay for traces that are improved by at least 25 Q20+ bases.
- A standalone version (LongTrace:Box) is available for users who wish to process there own traces in their own facility.
LongTrace DNA sequencing services
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 LongTrace DNA sequencing service uses a new approach to extract high quality sequence data from the poorly resolved regions of each trace. The LongTrace 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 ABI KB base caller to make significantly better base and quality calls on the trace. By combining the LongTrace raw data reprocessing with the KB base caller, the LongTrace DNA sequencing service is able to provide a completely seamless and straightforward method to increase sequence read length without requiring require sequence production protocol changes. An example of LongTrace processing is shown in Figure 1.
Figure 1. The KB v1.2 derived base and quality calls from an example ABI 3730xl 50cm capillary array sequence. A. The KB base called trace before LongTrace processing. B. The same KB base called trace after LongTrace processing. Note that the trace region shown is at base 1000.
The LongTrace reprocessing can be performed any time after the data is collected. This allows it to be used with previously collected data sets such as partial genome sequencing projects data. By applying LongTrace to the trace data from and reassembling the sequences the overall genome assembly can be significantly improved without the need to collect more sequence data. Additionally, as the output of the LongTrace service is a true ABI KB base called trace file, it means that LongTrace can be used with any KB compatible downstream software application. This feature allows LongTrace processing to be used with other base calling software such as phred or TraceTuner as required.
Features of the LongTrace DNA sequencing system
Simple to use
The LongTrace FTP DNA sequencing service is straightforward to use. After registering, you log in to our ftp server using any ftp program. You then select the ABI trace files for processing and upload them to your account on our ftp server. Files can be uploaded as individual files, zip archives, or folders of traces files. Once uploaded, the LongTrace service automatically reprocesses the trace files. When complete, an email is sent to you to let you know that the trace data set has been processed. You can then log in to your ftp account and download the improved traces (these are located in the "out" folder).
The LongTrace:Box system is equally easily to use. The system is based around a small, silent computer (an Apple Mac Mini running Windows XP Professional) and comes with an easy to use graphical software (Auto LongTrace) to allow the automatic processing of the traces simple as it can be used on any normal Windows file share network. In addition, the Auto LongTrace software provides trace improvement reports for both the individual user and the facility administrator making it straightforward to manage in a core lab environment.
LongTrace is compatible with trace files generated using the ABI BigDye™ v1.1 and v3.1 sequencing chemistries on the ABI 3730, ABI 3730xl, ABI 3130 or ABI 3100 DNA sequencing instruments for both the 36cm and 50cm capillary array lengths.
The LongTrace service can also be trial using the free LongTrace service. This service provides the exactly same read length as the paid ftp based service, however, you are limited to processing only 10 traces per day. If you have a large number of traces to process then we recommend that you register to use the high throughput ftp LongTrace service.
Increases trace read length and reduces errors
LongTrace 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. Distribution of read length improvement after LongTrace processing. Results from 3449 traces generated on an ABI3730xl 36cm capillary array.
Increases DNA sequencing throughput
LongTrace processing of 36cm capillary arrays traces provides read lengths equivalent to that obtained from standard 50cm arrays traces. Since standard 36cm array runs take only half the time of 50cm array runs, total sequencer throughput can be effectively doubled by the use of LongTrace. This enables users to avoid purchasing additional sequencing instruments as demand increases. In addition, Nucleics has developed a modified 36cm array run module that allows up to 28 runs per day (~51 min run time). This LongTrace optimized run module provides the same read length potential as the standard 2 hour 50cm array module, effectively permitting total sequencer throughput to be increased by more than 130%.
The improved DNA sequencing read lengths provided by LongTrace 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 LongTrace please or download the LongTrace DNA sequencing service or LongTrace:Box overview pdfs.