Comparison of Commercial Dye Terminator Removal Kits, Rev A
Julie Chang, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive,
Hercules, CA 94547
Introduction
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).
Methods
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 µl.
The cycling protocol was 95ºC for 3 min, followed by 35 cycles of 95°C
for 5 sec, 50ºC for 5 sec, and 60°C for 4 min. The final step was a 4°C
hold.
After cycling, all reactions were pooled and 20 µl aliquots of the pooled
reactions were purified using each of the dye terminator cleanup kits,
following the manufacturer’s 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
Evaluation Parameters
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 calling
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 retained 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 4–6 hr required to pack and
prepare the plate.
Conclusion
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 sequencing. 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.
Acknowledgements
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.
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