Introduction
LongTrace™ is a online DNA sequencing services 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 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 series of DNA sequencing instruments.
- A simple, easy to use ftp-based user interface that can be accessed using any ftp program.
- A "success only" charge. This means that you will only pay for traces that are improved by at least 25 Q20+ bases.
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 service
Simple to use
The LongTrace DNA sequencing service is straightforward to use. After collection of the trace data from the ABI instrument, a user logs in to our ftp server. The ABI trace files for processing are selected and uploaded to the ftp server using any rftp program. Once uploaded, the LongTrace software reprocesses then KB base calls the sequence files. When complete, an email is sent to the user informing them that the trace data set has been processed. The user logs in to their ftp account and downloads their improved traces which are now ready for use in other downstream applications.
LongTrace is compatible with trace files generated using the ABI BigDye™ v1.1 and v3.1 sequencing chemistries on the ABI 3730 and ABI 3730xl DNA sequencing instruments for both the 36cm and 50cm capillary array lengths. Recently, ABI 3130 and 3100 support has also been added.
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 overview pdf.