Julie Chang, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547
Capillary sequencing technology has altered the field of genomics, increasing throughput and automating the sequencing process. This high-throughput platform has become a standard in production laboratories, driving such achievements as the completion of the Human Genome Project.
One of the requirements for achieving this higherthroughput processing is high sample purity. Samples must be free of unincorporated dye terminators and other contaminants, including salt.
Achieving a High-Quality Sequence
Many factors besides sample cleanup contribute to a highquality sequence read. It is essential to start with a pure template that has been prepared using a reliable method. Poorly prepared template will result in poor sequencing results, including low signal strengths, short reads, and dye blob retention. It is also important to use the proper amounts of reagents in cycle sequencing reactions. Too much or too little template or primer may result in reads that are difficult to interpret and analyze. Another factor to be aware of is the use of optimized cycling parameters proper annealing temperatures, cycle times, cycle numbers, etc. Finally, instrument error can be minimized through proper matrix installation, fresh polymer, and clean and unclogged capillaries. These guidelines will help to increase the likelihood of a high-quality sequence.
Evaluation of Kits
Four parameters relevant to the evaluation of dye terminator cleanup kits will be discussed: signal intensity, Phred analysis, dye blob removal, and cost. A total of five 96- well dye terminator removal kits from various manufacturers were assessed using these four criteria (Table 1).
Cycle sequencing was performed using BigDye terminators, version 3.0 (Applied Biosystems). The plasmid template used was pGEM-3Zf(+), acquired from Promega. The primer used was a custom-ordered T7 primer (Operon). Half-reaction volumes (half of the recommended volume of terminator ready reaction mix) were employed, supplemented with a 5x buffer (10 mM MgCl2, 400 mM Tris, pH 9.0) to 20 l.
The cycling protocol was 95C for 3 min, followed by 35 cycles of 95C for 5 sec, 50C for 5 sec, and 60C for 4 min. The final step was a 4C hold.
After cycling, all reactions were pooled and 20 l aliquots of the pooled reactions were purified using each of the dye terminator cleanup kits, following the manufacturers protocol. Purified samples were dried down, resuspended in Hi-Di formamide (Applied Biosystems), injected into the ABI PRISM 3100 DNA sequencer for 20 sec, and run for 2.5 hr. Sequence data were analyzed using both the ABI PRISM sequence analysis software (version 3.3) and Phred software.
Results and Discussion
Signal intensity reflects both the efficiency of sample recovery and the purity of the sample. Sample recovery and purity are especially important in capillary sequencing, where even small amounts of contaminants can interfere with the uptake of sample into the capillary. In general, the higher the sample quantity and quality, the better the signal intensity and overall sequence quality.
Phred base-specific quality values reflect the accuracy of base ca lling and the reliability of a sequencing read. A quality value of q≥20 for an individual base reading indicates an accuracy of 99%. Phred scores, determined from the total number of bases having a score of q≥20, are synonymous with read lengths and are considered by large sequencing facilities to be the standard for determining the accuracy of a read.
Unincorporated dye terminators that are not removed from a sample prior to sequencing will show up as dye blobs. These dye blobs not only obscure data and interfere with base calling, but can lower overall Phred scores.
Cost is the final parameter to consider when selecting a sample purification kit. In high-throughput processes, small cost differences per sample add up to significant differences in overall operation costs.
Bio-Rad SEQueaky Kleen H2O Kit
The SEQueaky Kleen H2O dye terminator removal kit performed the best in all categories tested. At $0.70 per prep (Table 2), it is one of the least expensive prepacked kits on the market. With a handling time of just 4 min, it also performs the fastest purifications.
QIAGEN DyeEx 96 Kit
DyeEx 96 performance was the most similar to SEQueaky Kleen H2O in generating high signal intensities (Figure 1). However, the DyeEx 96 kit requires an additional wash and spin step prior to sample loading in order to achieve this signal. Although the cost per prep is the lowest of the prepacked kits that we tested, it did not match SEQueaky Kleen H2O in its ability to completely remove unincorporated dye terminators (Table 2).
Princeton Separations Centri-Sep Kit
Samples processed with the Centri-Sep kit retaine d a large number of dye blobs (Table 2) and gave lower signal intensities than samples processed with the SEQueaky Kleen H2O and DyeEx 96 kits. The most noticeable disadvantage of this kit was the high centrifugation speed required (1,500 x g), which can cause unnecessary wear on a centrifuge. Centri-Sep is also the most expensive kit tested, at a cost of $0.89 per prep.
Edge Biosystems Performa DTR 96 Kit
The Performa DTR 96 kit removed dye blobs as effectively as SEQueaky Kleen H2O; all of the other kits showed at least one dye blob per sample. However, Performa DTR 96 gave the lowest signal intensities and Phred scores (Figure 1). At $0.83 per prep, it is also one of the more expensive kits available.
Millipore MultiScreen-HV Plate and Sephadex Resin
Although the MultiScreen plate and resin protocol has the lowest cost per prep among the kits tested, it is also the most labor-intensive, requiring the user to fill the plate with resin, hydrate, rinse, and then proceed with purification. The extra user intervention and time commitment necessary slow down overall throughput. In addition, this kit retained the most dye blobs, contrary to its claim of complete removal of dye terminators. Lastly, the MultiScreen protocol had the longest purification time requirement. Whereas SEQueaky Kleen H2O purification is completed in 4 min, MultiScreen requires 10 min, in addition to the extra 46 hr required to pack and prepare the plate.
Sample cleanup prior to sequencing is essential to achieving high-quality sequence data. Sample purity is even more crucial for capillary sequencing than it is for slab gel sequencin g. Of the five kits compared, SEQueaky Kleen H2O and Performa DTR 96 were the only two that consistently removed dye blobs (Table 2). In addition, highthroughput requirements demand other criteria, such as convenience and acceptable cost. The SEQueaky Kleen H2O kit meets or exceeds the performance of comparable dye terminator removal kits. Giving high signal intensities, complete removal of dye terminators, and long read lengths, SEQueaky Kleen H2O offers the best quality at a cost-effective price.
Comparison of dye terminator removal kits and ABI PRISM 3100 DNA sequencing were performed by Davis Sequencing, LLC, Davis, CA, USA.
ABI PRISM, Big Dye, and Hi-Di are trademarks of Applera. pGEM is a trademark of Promega. Sephadex is a trademark of Amersham Biosciences.
back to top