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Vol. 11, Issue 1, 3-11, January 2001
REVIEW
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DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications.
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The development of DNA sequence determination
techniques with enhanced speed, sensitivity, and throughput are of
utmost importance for the study of biological systems. Conventional DNA
sequencing relies on the elegant principle of the dideoxy chain
termination technique first described more than two decades ago (Sanger
et al. 1977
). This multi-step principle has gone through major
improvements during the years to make it a robust technique that has
been used for the sequencing of several different bacterial, archeal,
and eucaryotic genomes (http://www.ncbi.nlm.nih.gov, and
http://www.tigr.org). However, this technique faces limitations in both
throughput and cost for most future applications. Many research groups
around the world have put effort into the development of alternative principles for DNA sequencing. Three methods that hold great promise are sequencing by hybridization (Bains and Smith 1988; Drmanac et al.
1989; Khrapko et al. 1989; Southern 1989), parallel signature sequencing based on ligation and cleavage (Brenner et al. 2000), and
pyrosequencing (Ronaghi et al. 1996, 1998b). Pyrosequencing has been
successful for both confirmatory sequencing and de novo sequencing.
This technique has not been used for genome sequencing due to the
limitation in the read length, but it has been employed for
applications such as genotyping (Ahmadian et al. 2000a; Alderborn et
al. 2000; Ekström et al. 2000; Nordström et al. 2000b),
resequencing of diseased genes (Garcia et al. 2000), and sequence
determination of difficult secondary DNA structure (Ronaghi et al.
1999). This article reviews the historical and technical aspects of the
technique with regards to general principles, different strategies,
application of the technique to different formats, and instrumentation.
The performance of the technique in different applications is also discussed.
Pyrosequencing
Pyrosequencing is a DNA sequencing technique that is based on the
detection of released pyrophosphate (PPi) during DNA synthesis. In a
cascade of enzymatic reactions, visible light is generated that is
proportional to the number of incorporated nucleotides (Fig.
1). The cascade starts with a nucleic acid
polymerization reaction in which inorganic PPi is released as a result
of nucleotide incorporation by polymerase. The released PPi is
subsequently converted to ATP by ATP sulfurylase, which provides the
energy to luciferase to oxidize luciferin and generate light. Because the added nucleotide is known, the sequence of the template can be
determined. The nucleic acid molecule can be either RNA or DNA.
However, because DNA polymerases show higher catalytic activity than
RNA polymerases for limited nucleotide extension, efforts have been
focused on the use of a primed DNA template for pyrosequencing. Standard pyrosequencing uses the Klenow fragment of Escherichia coli DNA Pol I, which is a relatively slow polymerase (Benkovic and
Cameron 1995). The ATP sulfurylase used in pyrosequencing is a
recombinant version from the yeast Saccharomyces cerevisiae (Karamohamed et al. 1999a) and the luciferase is from the American firefly Photinus pyralis. The overall reaction from
polymerization to light detection takes place within 3-4 sec at room
temperature. One pmol of DNA in a pyrosequencing reaction yields
6 × 1011 ATP molecules which, in turn, generate more
than 6 × 109 photons at a wavelength of 560 nanometers.
This amount of light is easily detected by a photodiode,
photomultiplier tube, or a charge-coupled device camera (CCD) camera.
There are two different pyrosequencing strategies that are currently
available: solid-phase pyrosequencing (Ronaghi et al. 1996) and
liquid-phase pyrosequencing (Ronaghi et al. 1998b). Solid-phase
pyrosequencing (Fig. 2) utilizes immobilized DNA in the three-enzyme system described previously. In
this system a washing step is performed to remove the excess substrate
after each nucleotide addition. In liquid-phase pyrosequencing (Fig.
3) apyrase, a nucleotide-degrading enzyme
from potato, is introduced to make a four-enzyme system. Addition of
this enzyme has eliminated the need for solid support and intermediate
washing thereby enabling the pyrosequencing reaction to be performed in a single tube. These formats are described in detail in this review.
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History
The theory behind sequencing-by-synthesis was described in 1985 (Melamede 1985) and based on this principle, detection of pyrophosphate
was used in DNA sequencing (Hyman 1988). Efforts were also put into the
development of this principle for sequence determination using labeled
nucleotides (Canard and Sarfati 1994; Cheesman 1994; Metzker et al.
1994; Rosenthal 1989; Tsien et al. 1991). However, Metzker et al.
(1994) showed that the incorporation efficiency of labeled nucleotides
is low, causing nonsynchronized extension, which made it difficult to
sequence more than a few bases. Synchronized extension in
sequencing-by-synthesis requires exonuclease-deficient
(exo
) DNA polymerase and unmodified nucleotides. We used
coupled enzymatic reactions, which were used earlier to assay
polymerase activity (Nyren 1987) to monitor stepwise DNA synthesis
using exopolymerase and unlabeled nucleotides
(pyrosequencing). However, false signals were always observed when dATP
was added into the pyrosequencing solution (Ronaghi et al. 1996). The
first major improvement was substitution of dATP
S for dATP in the
polymerization reaction, which enabled the pyrosequencing reaction to
be performed in homogeneous phase in real time (Ronaghi et al. 1996).
It was later shown that the nonspecific signals were attributed to the fact that dATP is a substrate for luciferase. Conversely, dATPS was found to be inert for luciferase, yet could be incorporated efficiently by all DNA polymerases tested (Ronaghi et al. 1996). This
strategy was used successfully for sequencing of PCR-generated DNA
material (Ronaghi et al. 1996).
The second improvement was the introduction of apyrase to the reaction
to make a four-enzyme system (Ronaghi et al. 1998b). The addition of
apyrase allowed nucleotides to be added sequentially without any
intermediate washing step. This enzyme shows high catalytic activity
and low amounts of this enzyme in the pyrosequencing reaction system
efficiently degrade the unincorporated nucleoside triphosphates to
nucleoside diphosphates and subsequently to nucleoside monophosphate.
Apyrase is less inhibited by its products as compared to other
nucleotide-degrading enzymes.
Most recently, the addition of ssDNA-binding protein to the
pyrosequencing reaction system has simplified the optimization of
different parameters in pyrosequencing. This protein has proven to be
useful for long read sequencing and sequencing of difficult templates,
as well as providing flexibility in primer design (Ronaghi 2000).
Template Preparation for Pyrosequencing
Template preparation for pyrosequencing is straightforward. After generation of the template by PCR, the product should be purified prior to pyrosequencing. Unincorporated nucleotides and PCR primers in PCR reaction perturb the pyrosequencing reaction. The salt in the PCR reaction slightly inhibits the enzyme system and should be removed or diluted. Two strategies currently available for generation of a primed DNA template for pyrosequencing are described below.
Solid-Phase Template Preparation
Streptavidin-coated magnetic beads have been used to prepare primed DNA template for pyrosequencing. This technology enables biotinylated PCR product to be captured onto magnetic beads. After sedimentation, the remaining components of the PCR reaction can be removed by washing to obtain pure double-stranded DNA followed by alkali denaturation to yield ssDNA. Both the immobilized biotinylated and nonbiotinylated strands in solution can be used as pyrosequencing templates (Ronaghi et al. 1998a, 1999). This template preparation system has given
high-quality sequence data with low background signals.
Enzymatic Template Preparation
Recently, enzymatic template preparation was developed for sequencing on double-stranded DNA template (Nordstrom et al. 2000a,b). This template preparation method employs a nucleotide-degrading enzyme
and exonuclease I. The enzymes are added to the PCR product and the
mixture is incubated at room temperature or 35°C. During this step,
the nucleotide-degrading enzyme removes the nucleotides and exonuclease
I degrades the PCR primers remaining from the amplification step. The
sequencing primer is dispensed into the treated mixture and the
temperature of the solution is increased to heat-inactivate the
enzymes. Template/primer complexes are formed by rapid cooling of the
solution. Two different enzyme systems can be used. The use of alkaline
phosphatase from shrimp or calf intestine together with exonuclease I
allows the template to be prepared within 20 min, whereas a combination
of a low amount of apyrase, inorganic PPi, and exonuclease I enables
the template to be prepared in three min. High quality pyrosequencing
data has been obtained by enzymatic template preparation using a
prototype pyrosequencing system that employs a very sensitive light
detector. However, this template preparation method needs to be further optimized for use with the standard system that uses microtiter plates,
because the dilution that is required to compensate for incompatible
buffer systems results in low amounts of primed DNA template.
Improvements may be obtained by running PCR in a compatible buffer with
pyrosequencing reaction or by using a more sensitive CCD camera in the
pyrosequencing machine.
Pyrosequencing Enzyme Systems
Pyrosequencing takes advantage of the cooperativity of several
enzymes to monitor DNA synthesis. Parameters such as stability, fidelity, specificity, sensitivity, KM, and
kcat (Table 1) are of
utmost importance for the optimal performance of the enzymes used in
the reaction (Ronaghi 1998). The kinetics of the enzymes can be studied
in real time by following the pyrosequencing signals (a pyrogram). The
slope of the ascending curve in a pyrogram (Figs. 4 and
6)
is determined mainly by the activities of polymerase and ATP
sulfurylase; the height of the signal is determined by the activity of
luciferase, and the slope of the descending curve by the efficiency of
nucleotide removal. In the solid-phase system using microfluidics,
which employs the three-enzyme system, the descending curve is
determined by the washing efficiency. In the four-enzyme system of
liquid-phase pyrosequencing, the accumulation of inhibitory substances
decreases the efficiency of luciferase and apyrase. In both systems,
the activity of ATP sulfurylase is relatively constant during the
sequencing reaction. In pyrosequencing, the most critical reactions are
DNA polymerization and nucleotide removal by either washing or
enzymatic degradation. Nucleotide removal (descending curve) competes
with the polymerization reaction (ascending curve). Therefore, slight
changes in the kinetics of these reactions directly influence the
performance of the sequencing reaction.
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Polymerization Reaction
An excess amount of DNA polymerase relative to DNA template in the pyrosequencing reaction ensures that the primed DNA template is bound efficiently by polymerase and that, at the time of nucleotide addition, polymerization takes place immediately. To obtain rapid polymerization, the nucleotide concentration must be above the KM of the DNA polymerase (Table 1). Conversely, if the concentration of the nucleotides is too high, lower fidelity of the polymerase is observed (Eckert and Kunkel 1990; Cline et al. 1996), even though the
KM for misincorporation is much higher than that of correct incorporation (Gillin and Nossal 1976; Topal et al. 1980; Capson et al. 1992). A high fidelity can be achieved by using polymerases with inherent exonuclease activity, however, this has the
disadvantage that primer degradation can occur causing out of phase
signals. Although the exonuclease activity of Klenow polymerase is
relatively low, it has been found that the 3' end of the primer was
degraded during long incubations in the absence of nucleotides. Even
without exonuclease activity, an induced-fit binding mechanism in the
polymerization step (Wong et al. 1991) provides a very high selectivity
for the correct nucleotide, with a fidelity of
105-106 when the nucleotide are added slightly
above the KM. In pyrosequencing, exo-polymerases,
such as exo-Klenow or Sequenase, catalyze the incorporation of a
nucleotide only in the presence of a complementary nucleotide,
confirming the high fidelity of these enzymes even in the absence of
proof-reading exonuclease activity. The KM and kcat for one-base incorporation is lower than that
for the incorporation of several bases for most polymerases (Van
Draanen et al. 1992). However, the KM values for
nucleotides are much lower for DNA polymerases than for apyrase (Table
1). Therefore, an increased fidelity in the system can be obtained as
the nucleotide concentration necessary for efficient polymerization is
relatively low, and apyrase degrades nucleotides to a concentration far
below the KM of the polymerase in less than five sec.
Detection Enzymes
On successful polymerization, a proportional amount of PPi is released. ATP sulfurylase converts PPi to ATP in ~1.5 sec and the generation of light by luciferase takes place in <0.2 sec. In the four-enzyme system accumulation of AMP and dAMPS inhibits the
luciferase activity. Kinetics of the enzymes in the detection reaction
can be followed by the addition of a known amount of PPi to the
pyrosequencing enzyme system.
Nucleotide Removal
To allow iterative addition, nucleotides must be removed from the pyrosequencing reaction. In the four-enzyme system, nucleotides are removed enzymatically. The nucleotide-degrading enzyme must possess the following properties: First, the enzyme must hydrolyze all deoxynucleoside triphosphates at approximately the same rate; second, it must hydrolyze ATP to prevent the accumulation of ATP between cycles; third, the time for nucleotide degradation by the nucleotide-degrading enzyme must be slower than nucleotide incorporation by the polymerase. It is also important that the yield of primer-directed incorporation is as close to 100% as possible before the nucleotide-degrading enzyme has degraded the nucleotide to a concentration below the KM of the polymerase (Table 1). Pyrograms obtained from liquid-phase pyrosequencing (Fig. 4) show that apyrase fulfils the criteria described above. A constant signal intensity for each base incorporation is obtained during the course of a reaction determining high efficiency of this system (Fig. 4). In solid-phase pyrosequencing (three-enzyme system), washing in a controlled manner should show the same advantages offered by apyrase in the four-enzyme system. An additional advantage of the three-enzyme system is that no accumulation of inhibitory substances will be observed, because washing is performed between each nucleotide addition.Extending the Read Length of Pyrosequencing
For many applications such as genome sequencing and gene sequencing,
a long read is desirable. Several criteria must be met to obtain a long
read length in pyrosequencing: (1) The enzyme system must be stable;
(2) there must be a low misincorporation; and (3) nucleotide extension
must be synchronized. The enzyme system has been shown to be stable in
its buffer system during the sequencing reaction as relatively constant
signal intensity is obtained for each individual nucleotide (Fig. 4).
In the four-enzyme system, removal of inhibitory substances from the
reaction and minimizing the dilution effect gives rise to 200 nucleotide reads (Ronaghi et al. 2000). The use of unlabeled
nucleotides, addition of nucleotide in a concentration slightly above
the KM, and rapid removal of nucleotides from the
solution increases the fidelity of DNA synthesis. Although a relatively
high signal-to-noise ratio is obtained in pyrosequencing,
misincorporation may play an important role in limiting longer reads.
Possible misincorporation terminates the primer strands, which results
in decreased signal intensity in the course of a reaction.
Nonsynchronized extensions are either a result of minus frame shift
(when some of the primer strands get one, or a few, nucleotides behind
the other synchronized primer strands during extension) or plus frame
shift (when some of the primer strands get one, or a few, nucleotides
ahead of other synchronized primer strands during extension). Using
exo DNA polymerase reduces the minus frame shift.
Insufficient exposure of nucleotides to polymerase can cause minus
frame shift, which is sometimes observed in long homopolymeric regions.
In the four-enzyme system of pyrosequencing, apyrase degrades the
nucleotides below the KM for polymerase, not
allowing enough time for the polymerase to complete the polymerization
of these regions. The use of lower amounts of apyrase, or a second
addition of the same nucleotide, solves this problem. Improvements can
also be obtained by the use of ssDNA-binding protein in the
pyrosequencing reaction solution (Ronaghi 2000). In a microfluidic
format, complete incorporation can be controlled easily by a delay in
washing. Plus frame shift is mainly a problem for the four-enzyme
system of pyrosequencing and normally is caused by enzyme contaminants
or inefficient nucleotide degradation. A contaminating enzyme such as
nucleoside diphosphate kinase, which normally is found in commercially
available ATP sulfurylase and apyrase, converts the nucleoside
diphosphate to nucleoside triphosphate (Karamohamed et al. 1999b), a
substrate for polymerase. Another parameter causing plus frame shift is inefficient degradation of nucleotides by apyrase and usually is seen
in later cycles of pyrosequencing due to the accumulation of inhibitory
substances. Further works are underway to remove the inhibitory
substances from the reaction system either by purification or enzymatic
degradation. Another factor reducing the efficiency of the four-enzyme
system is the dilution effect. In the four-enzyme system, the
nucleotides are iteratively added to the pyrosequencing solution
thereby increasing the reaction volume at each step. Although the
volume of nucleotides added is as little as 200 nanoliter/min, dilution
can be seen in long-read sequencing. Dilution lowers the enzyme
concentrations thereby decreasing their efficiency. Possible
improvements include reducing the volume of nucleotide delivery or
running the reaction at higher temperatures to increase evaporation.
Challenges for Pyrosequencing Technology
An inherent problem with the described method is de novo sequencing
of polymorphic regions in heterozygous DNA material. In most cases, it
will be possible to detect the polymorphism. If the polymorphism is a
substitution, it will be possible to obtain a synchronized extension
after the substituted nucleotide. If the polymorphism is a deletion or
insertion of the same kind as the adjacent nucleotide on the DNA
template, the sequence after the polymorphism will be synchronized.
However, if the polymorphism is a deletion or insertion of another
type, the sequencing reaction can become out of phase, making the
interpretation of the subsequent sequence difficult. If the
polymorphism is known, it is always possible to use programmed
nucleotide delivery to keep the extension of different alleles
synchronized after the polymorphic region. It is also possible to use a
bidirectional approach (Ronaghi et al. 1999) whereby the complementary
strand is sequenced to decipher the sequence flanking the polymorphism.
Another inherent problem is the difficulty in determining the number of
incorporated nucleotides in homopolymeric regions, due to the nonlinear
light response following incorporation of more than 5-6 identical
nucleotides. The polymerization efficiency over homopolymeric regions
has been investigated and the results indicate that it is possible to
incorporate 
10 identical adjacent nucleotides in the presence of
apyrase (Ronaghi 2000). However, to elucidate the correct number of
incorporated nucleotides, it may be necessary to use specific software
algorithms that integrate the signals. For resequencing, it is possible
to add the nucleotide twice for a homopolymeric region to ensure
complete polymerization.
Applications of Pyrosequencing
Pyrosequencing has opened up new possibilities for performing sequence-based DNA analysis. The availability of an automated system for liquid-phase pyrosequencing (PSQ 96 system, http://www.pyrosequencing.com) has allowed the technique to be adapted for high-throughput analyses. This section describes some of the potential applications of pyrosequencing.
Genotyping of Single-Nucleotide Polymorphisms
For analysis of single-nucleotide polymorphisms (SNPs) by pyrosequencing, the 3'-end of a primer is designed to hybridize one or a few bases before the polymorphic position. In a single tube, all the different variations can be determined as the region is sequenced. A striking feature of pyrogram readouts for SNP analysis is the clear distinction between the various genotypes; each allele combination (homozygous or heterozygous) will give a specific pattern compared with the two other variants (Ahmadian et al. 2000a; Alderborn et al. 2000;
Ekstrom et al. 2000; Nordstrom et al. 2000b). This feature makes typing
extremely accurate and easy. Relative standard deviation values for the
ratio between key peaks of the respective SNPs and reference
counterparts are 0.1 (Alderborn et al. 2000). Simple manual
comparison of predicted SNP patterns and the raw data obtained from the
PSQ 96 system can score an SNP, especially as no editing is needed.
Because specific patterns can be readily achieved for the individual
SNPs, it will also be possible to automatically score the allelic
status by pattern recognition software. In a study based on results
from three different laboratories, 26 different SNPs and >1600 DNA samples were analyzed. The algorithm classified the data from 94% of
the samples as good or medium quality and 99.4% of these were
automatically assigned the expected genotypes (B. Ekström, pers.
comm.). The major reason for low quality data was insufficient signal/noise typically caused by low efficiency in PCR amplification. As pyrosequencing signals are very quantitative, it is possible to use
this strategy for the studies of allelic frequency in large population.
This system allows >5000 samples to be analyzed in 8 h. Furthermore,
pyrosequencing enables determination of the phase of SNPs when they are
in the vicinity of each other allowing the detection of haplotypes
(Ahmadian et al. 2000b).
Microbial Typing
DNA markers used for typing normally contain both conserved and variable regions. A DNA primer complementary to the conserved or semiconserved region is usually employed to sequence the variable region. In bacteria, 16S rRNA gene is commonly used to identify different species and strains. By analyzing a sequence between 20-100 nucleotides on 16S rRNA gene, it is possible to taxonomically group different bacteria and, in many cases it is possible to get information about the strains. Pyrosequencing is now being applied for rapid typing of large number of bacteria, yeasts, and viruses (B. Gharizadeh, pers. comm.).Resequencing
Pyrosequencing is currently the fastest method for sequencing a PCR product. Because pyrosequencing generates an accurate quantification of the mutated nucleotides, the resequencing of PCR-amplified disease genes for mutation scanning will be one of the more interesting applications. Using this technique for resequencing results in longer read length than de novo sequencing because nucleotide delivery can be specified according to the order of the sequence. Programmed dispensing generates a signal for each addition in a pyrogram, therefore variation in the pattern indicates the appearance of a mutation. This strategy has been used for resequencing of the p53 tumor suppressor gene where mutations were successfully determined and quantified (Garcia et al. 2000).
Tag Sequencing
The sequence order of nucleotides determines the nature of the DNA. Theoretically, eight or nine nucleotides in a row should define a unique sequence for every gene in the human genome. However, it has been found that to uniquely identify a gene from a complex organism such as human, a longer sequence of DNA is needed. In a pilot study, it was found that 98% of genes in a human cDNA library could be uniquely identified by sequencing a length of 30 nucleotides. Pyrosequencing was used to sequence this length for gene identification from a human cDNA library and the results were in complete agreement with longer sequence data obtained by Sanger DNA sequencing. Pyrosequencing offers high-throughput analysis of cDNA libraries because 96 samples can be analyzed in less than one hour. Like Sanger DNA sequencing, pyrosequencing also has the advantage of library screening, as the original cDNA clone is directly available for further analysis.Analysis of Difficult Secondary Structures
Hairpin structures are common features in genomic material and have been proposed to have regulatory functions in gene transcription and replication. However, analyzing these sequences by conventional DNA sequencing usually gives rise to DNA sequence ambiguities seen as "run-off" or compressions. These problems have been associated with gel electrophoresis. Pyrosequencing was successfully applied to decipher the sequence of such regions (Ronaghi et al. 1999). Klenow DNA
polymerase was used in these studies in which relatively high
strand-displacement activity in reading through these structures was shown.
Instrumentation
Automation Based on Microtiter Plate Format
An automated version of a pyrosequencing machine was recently developed (http://www.pyrosequencing.com). The automated version uses a disposable inkjet cartridge for precise delivery of small volume (200 nL) of six different reagents into a temperature-controlled microtiter plate (Fig. 5). The microtiter plate is under continuous agitation to increase the rate of the reactions. A lens array is used to efficiently focus the generated luminescence from each individual well of the microtiter plate onto the chip of a CCD camera. Nucleotides are dispensed into alternating wells with a delay to minimize the crosstalk of generated light between different wells. A cooled CCD camera images the plate every second to follow the exact process of the pyrosequencing reaction. Data acquisition modules and an interface for PC connection are used in this instrument. Software running under Windows NT enables individual control of the dispensing order for each well. Prior to pyrosequencing, the reagents and each of the four nucleotides are loaded into the inkjet cartridge that is mounted in the instrument. A microtiter plate containing primed DNA template is placed into the pyrosequencing machine, and after the enzymes and substrate have been delivered by the inkjet, nucleotides are added to the solution according to the specified order. The signals in a pyrogram (Fig. 4) show high quality sequence data with high signal-to-noise ratio with the height of the peaks proportional to the number of incorporated nucleotides. A high-throughput version of this machine is also under development, which will allow the analysis of50,000
SNPs per day (B. Ekström, pers. comm.).
Microfluidics Using Solid-Phase Pyrosequencing
Running pyrosequencing on solid-phase in a microfluidic system offers several advantages compared with other described formats: (1) Sequencing can be performed faster because the cycling time for each nucleotide addition can be reduced; (2) there is no accumulation of inhibitory substances because washing is performed after each cycle; (3) it is possible to use lower amounts of enzymes to reduce the cost, and (4) integration of PCR amplification, template preparation, and pyrosequencing analysis in a single flow system can be envisaged. Using this format, DNA templates are immobilized on a solid support that enables iterative washing. Eventually it may be possible to immobilize the detection enzymes (ATP sulfurylase and luciferase) onto a solid support to further reduce enzyme consumption. Pyrosequencing in a microfluidic format has the potential to be used for very long reads because no accumulation of inhibitory substances will be obtained. Different microfluidic formats are currently being tested for pyrosequencing analysis. The pyrogram in Figure 6 demonstrates promising sequencing data with a relatively high signal-to-noise ratio that was obtained in a centrifugal force-driven compact disc microfluidic device (Eckersten et al. 2000).
Array Pyrosequencing
Developments in microarray technology have opened new possibilities for sequence-based analysis. The major advantages of these formats are low cost and high throughput. Pyrosequencing can be applied on both ordered and random arrays. In the latter array format, pyrosequencing data provides high amounts of information to reveal the sequence of the DNA template to be analyzed, eliminating the decoding step. A similar strategy can be applied to tag sequencing of a cDNA library immobilized on a solid surface using a common sequencing primer. A system has been built that employs a nucleotide delivery module, a DNA array, and a CCD camera (M. Ronaghi N. Pourmand, M. Jain, T. Willis, and R.W. Davis, in prep.). A piezoelectric ultrasonic sprayer was recently developed to enable homogenous delivery of nucleotide onto a microarray. A single sprayer is used to deliver all four different nucleotides, with washing of the nozzle between each delivery. Data from single-base extension on an oligonucleotide template attached to a glass slide has been obtained, showing the feasibility of the pyrosequencing enzymatic reactions in this format. It should be noted that in this chemiluminescence assay the energy available for detection is proportional to the amount of template in the reaction, whereas in fluorescence assay the energy available for detection can be increased through the use of more powerful lasers. Consequently, sensitive detection systems must be employed to allow detection of miniature pyrosequencing reaction. Imaging a pyrosequencing reaction onto currently available CCD technology, we believe that the smallest detectable reaction should contain
5000 template molecules. Further
optimization needs to be performed in terms of diffusion and
variability of the amount of available primed DNA templates before
application of such a format for reliable high throughput DNA sequencing.
Software for Pyrogram Analysis
Specialized software for SNP analysis of pyrograms obtained from liquid-phase pyrosequencing has been developed (http://www.pyrosequencing.com). This software enables analyses of selected wells in a microtiter plate and automatically performs genotyping as well as quality assessment of the raw data utilizing a novel SNP genotyping algorithm. Based on pattern recognition, this algorithm automatically scores the genotype and calculates a quality value for each SNP scored. The assignment of quality values is based on a number of different parameters, including difference in match between the best and next best choice of genotypes, agreement between expected and obtained sequence around the SNP, signal-to-noise ratios, variance in peak heights around the SNP, and peak width. This software has also been used for other applications such as EST sequencing, microbial typing, and confirmatory sequencing, however, until now the base-calling has been performed manually. Specialized software for pyrosequencing of longer reads is currently under development with automatic base-calling (B. Ekström, pers. comm.).
Future Trends
Genome sequencing provides tremendous amounts of information that can be used in several different areas of biology. Comparative sequencing will dominate DNA sequencing to identify variations across those genomes that have been sequenced. Technologies with high accuracy for the identification of these variations in genome-wide scanning will have great value. Pyrosequencing has shown excellent accuracy for analysis of polymorphic DNA fragments. This technology has also been used for quantification of allelic frequency in populations. While the variations are characterized, correlation of variation to phenotype can be performed. Pyrosequencing will have a large impact in that area because a large number of samples can be pooled in one pyrosequencing reaction. A high throughput version of this technology can potentially be used for resequencing of genomes.
Pyrosequencing technology is relatively new and there is much room for developments in both chemistry and in instrumentation. The technology is already time- and cost-competitive (the cost is currently 69 cents per sample using standard pyrosequencing; http://www.pyrosequencing.com) when compared with the existing sequencing methods. Work is underway to further improve the chemistry, to measure the sequencing efficiency at elevated temperatures, and to run the reaction in miniaturized formats. The advantage of pyrosequencing in miniaturized formats may lie in the ease with which large numbers of high-density arrays can be manufactured and the future integration of sample preparation with these devices. Success in miniaturization of this technique into high density microtiter plates, microarrays, or microfluidics will reduce the cost and increase the throughput by one to two orders of magnitude, a crucial step for large scale genetic testing.
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ACKNOWLEDGMENTS |
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The author is supported by an NIH grant. I thank Ronald Davis for valuable discussions, Guri Giaever, Joakim Lundeberg, Paul Hardenbol, Thomas Willis, Pål Nyrén, and Björn Ekström for valuable comments on this manuscript. I also thank Baback Gharizadeh, Afshin Ahmadian, Nader Pourmand, and Nigel Tooke for sharing their results on pyrosequencing.
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FOOTNOTES |
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E-MAIL mostafa{at}stanford.edu; FAX (650) 812-1975.
Article and publication are at www.genome.org/cgi/doi/10.1101/gr.150601.
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M. Haanpera, J. Jalava, P. Huovinen, O. Meurman, and K. Rantakokko-Jalava Identification of Alpha-Hemolytic Streptococci by Pyrosequencing the 16S rRNA Gene and by Use of VITEK 2 J. Clin. Microbiol., March 1, 2007; 45(3): 762 - 770. [Abstract] [Full Text] [PDF]
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