CounterTrace™ is a novel software and reagent designed to significantly improve the read length and overall quality when sequencing DNA on the ABI 3700 and 3730 DNA sequencers. The CounterTrace DNA sequencing system offers the following features:
- Up to 25% more phred Q20 bases from each trace
- Compatibility with the ABI 3700 and 3730 DNA sequencing instruments
- Easy to use in both large and small scale DNA sequencing centers
Overview of the CounterTrace DNA sequencing reagent & software
Current capillary DNA sequencer can generate sequences longer than 1100 bases, however, the reads usually have poorly resolved peaks after base 800. This latter trace region tends to have low quality scores and very large numbers of error.
CounterTrace uses an internal calibration standard to allow accurate measurement and processing of each sequencing trace (Figure 1). The CounterTrace additive contains DNA fragments of known size labeled with a proprietary fluorophore (CounterTrace standard). This fluorophore has been designed to enable its specific detection in four channel sequence data. Addition of the CounterTrace standard to each sequencing reaction after clean-up provides unique ‘landmark’ sizing peaks in the raw trace data. The CounterTrace DNA sequencing software uses the location of the standard peaks to enable accurate processing of the trace data. After removal of the standard peaks from the trace the CounterTrace DNA sequencing software output sequencing trace files in either abi or scf format. The sequencing trace file are base called and quality assigned using any user-defined software (e.g. Phred, LifeTrace, etc).
Figure 1. Overview of the CounterTrace DNA sequencing data processing
Features & benefits of the CounterTrace DNA sequencing system
Easy implementation in DNA sequencing core facilities
The CounterTrace DNA sequencing system is extremely simple to implement into any DNA sequencing facility. The CounterTrace additive is diluted to the appropriate concentration in the sequencer loading solution used by the facility (e.g. water, 50 µM EDTA, or formamide), and the sequencing sample processed as normal. After collection of the trace data, the CounterTrace software is used to reprocess the traces before base and quality calling with an external sequencing software package (e.g. phred). The CounterTrace software includes an automation feature that allows seamless integration into existing data pipelines.
CounterTrace is compatible with all common sequencing reagents and has been adopted for the common DNA sequencer models ABI 3700 and ABI 3730 for both the 36 and 50 cm capillary array lengths.
Increase in DNA sequencing trace read length
The CounterTrace software allows poorly resolved regions towards the end of the sequencing trace to be accurately processed, and thus provides the user with significantly longer and higher quality reads (Figure 2).
Figure 2. PHRED derived base and quality calls from an ABI3730 50 cm capillary array DNA sequence trace. A. Before CounterTrace processing. B. After CounterTrace processing.
CounterTrace offers increases in phred Q20 read lengths of up to 45% for individual traces, with an average increase of over 20% in high throughput production DNA sequencing environments (Figure 3).
Figure 3. Distribution of phred20 sequencing read lengths before (ABI) and after CounterTrace processing (CounterTrace). Results from 3840 DNA sequencing traces analyzed on a ABI3730xl 36 cm capillary array.
Increased DNA sequencing throughput
CounterTrace processed DNA sequencing traces run on the ABI 3730xl 36cm capillaries provided read lengths previously observed only with 50cm arrays (Figure 3). As these short-array runs take only half the time required for the longer arrays, the throughput can be effectively doubled with CounterTrace without the capital cost involved in purchasing additional instruments.
The improved read lengths provided by CounterTrace increases the throughput and lowers the per-base cost. In addition, these longer reads allow for DNA sequencing projects (eg. whole genome, cDNA clones) to be finished at lower coverage levels, offering further cost and time savings.
CounterTrace Price & Ordering Information