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Pyrosequencing

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Pyrosequencing

November 22, 2021 0

In 1993, one of the first second-generation DNA sequencers emerged, as Dr. Bertil Pettersson, Dr. Mathias Uhlen, and Pal Nyren introduced pyrosequencing, often described as the pioneer of next-generation DNA sequencing.1 Pyrosequencing is a gel-free and sequencing-by-synthesis DNA sequencing technology, which involves obtaining genetic information by synthesizing the elongating strand of DNA using the complementary strand as a template.1 

Pyrosequencing DNA Library Preparation

Sequencing begins with the fragmentation and denaturation of the DNA being sequenced to form fragments of single-stranded DNA (ssDNA) that are 300-500 nucleotides long, where adapter sequences are added to both ends (Figure 1A).2 Then, the resulting DNA library is covalently attached to microscopic beads via the adapter sequence which undergoes an emulsion polymerase chain reaction (emPCR) to form millions of identical copies of ssDNA covalently bound to microscopic beads (Figure 1B).2 These microscopic beads are added to a reaction plate, where there is only one bead in each well. Each of these wells contains DNA polymerase (DNAP), adenosine phosphosulfate (APS), ATP sulfurylase, luciferin, and luciferase.2 To initiate DNA synthesis, a primer complementary to the adapter sequence is added to the wells, which allows DNAP to bind and synthesize the elongating strand following the addition of one of the four deoxyribonucleotide triphosphates (dNTPs). It is important to note that rather than using deoxyadenosine triphosphate (dATP), deoxyadenosine alpha-thio triphosphate (dATPαS) is used instead, as it can be efficiently used by DNAP for incorporation into the elongating DNA strand without being recognized by luciferase.2 dATP can be recognized by luciferase, which would result in the unwanted production of light.2 

Pyrosequencing DNA library preparation process.

Figure 1: Pyrosequencing DNA library preparation process. A. Fragmentation and denaturation of the DNA being sequenced and the additional adapters and biotin. Individual attachment of ssDNA onto streptavidin-coated beads. B. emPCR and loading of reagents onto the reaction plate for pyrosequencing.

Pyrosequencing & Data Analysis

Each time a nucleotide is successfully incorporated into the elongating DNA strand, a pyrophosphate (PPi) is released (Figure 2A), which reacts with APS in the solution, resulting in the formation of ATP, which is catalyzed by ATP sulfurylase (Figure 2B).2,3 Then, the ATP drives the conversion of luciferin to oxyluciferin and light, which is catalyzed by luciferase. The resulting light generated is captured by the charged couple device sensor beneath the wells. The amount of light produced is directly proportional to the amount of ATP in the solution – which provides information about how many dNTPs were consecutively added to the elongating DNA strand (Figure 2C).3,4 However, this is one of the disadvantages of pyrosequencing as it is difficult to accurately identify and interpret homopolymer sequences, that are essentially many repeated nucleotides, as the signal becomes less clear.2,4 The process of adding one of the four deoxynucleotide triphosphates (dNTPs) until DNAP incorporates it into the elongating DNA strand is indicated via the detection of light and is repeated until it reaches the end of the DNA fragment. Once elongation is complete, the data is analyzed by computing programs, allowing scientists to successfully sequence the original DNA molecule. 

Pyrosequencing mechanism of action.

Figure 2: Pyrosequencing. A. With the incorporation of the appropriate nucleotide to the elongating strand, a PPi is released. B. The PPi reacts with APS to form ATP, and ATP enables luciferin to be converted to oxyluciferin and the release of light, which are catalyzed by ATP sulfurylase and luciferase, respectively. C. The emission of light is detected, allowing for the sequencing data to be collected. 

DNA Methylation Detection Capabilities

Understanding DNA methylation patterns is crucial for understanding gene expression, as the methylation of certain nucleotides may be responsible for the over or under-expression of crucial genes involved with disease.2,4 Pyrosequencing technologies have the added benefit of being able to detect these DNA methylation patterns. To do this, DNA sequencing occurs twice, with one batch of DNA undergoing an additional step prior to emPCR, called bisulfite conversion.2,4 During this process, the DNA fragments are treated with sodium bisulfite which results in the deamination of unmethylated cytosines into uracils, while methylated cytosines remain unchanged (Figure 3).4 Then, following emPCR, uracils are converted into thymines – where the resulting beads containing these sequences undergo pyrosequencing, as described earlier. Next, the resulting sequence is compared to the sequence of the DNA that did not undergo bisulfite conversion. By determining all the nucleotides where thymine is detected in the bisulfite converted DNA where a cytosine is detected in the original DNA sequence, the methylation pattern can be obtained.4

Pyrosequencing DNA methylation process.

Figure 3: Pyrosequencing DNA methylation library preparation. Bisulfite conversion causes all non-methylated cytosines to be converted into uracil. The uracils are then converted into thymines during PCR amplification, which allows researchers to obtain methylation patterns by comparing the bisulfite converted and non-bisulfite converted sequences.

Automation of Pyrosequencing Technology

Although the core technology described earlier was a large improvement over existing methods of sequencing at the time, there was still room for improvement. These advances occurred in 1998, when Dr. Mostafa Ronaghi, Dr. Mathias Uhlen, and Pal Nyren added an additional enzyme called apyrase into the reaction sample during the sequencing step.3,5 Apyrase is able to remove nucleotides that were not incorporated into the elongating strand by DNAP.5 From this modification, the two critical enzymes, DNAP and luciferase, in the light-emitting reaction remained in the solution throughout the elongation reaction. In addition to this, the entire reaction setup changed when the technology was transferred to Jonathan Rothberg and colleagues at 454 Life Sciences, where the DNA was tethered to a solid support, allowing the sequencing to occur in a massively parallel and high-throughput manner.6 As a result of this setup, pyrosequencing became the first DNA sequencing technology to successfully reach commercial markets as the 454 Roche sequencing system, following the acquisition of 454 Life Sciences in 2007.

Discontinuation of Pyrosequencing & Impact on the Future

Despite pyrosequencing being a powerful sequencing platform at the time, being widely used for mutation gene analysis, microbial identification, resistance typing, and epigenetic analysis, the technology has been discontinued in 2013.6,7 This is because the pyrosequencing platform turned obsolete and non-competitive following the introduction of more advanced and rapid DNA sequencing technology. Regardless, pyrosequencing has served as a pivotal innovation in this space, paving the way for the subsequent generations of DNA sequencing platforms. As a result, DNA sequencing technologies today can provide enormous amounts of information at a fraction of the time – enabling vast changes in our understanding of disease, how to treat it, and patient outcomes.

References

1. Nyren P, Pettersson B, Uhlen M. Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay. Anal Biochem. 1993;208(1):171-175.

2. Kreutz M, Schock G, Kaiser J, Hochstein N, Peist R. PyroMark® Instruments, Chemistry, and Software for Pyrosequencing® Analysis. Methods. Mol. Biol. 2015:17-27.

3. Ronaghi M, Uhlén M, Nyrén P. A Sequencing Method Based on Real-Time Pyrophosphate. Science. 1998;281(5375):363-365.

4. Tost J, Gut I. DNA methylation analysis by pyrosequencing. Nat. Protoc. 2007;2(9):2265-2275.

5. Ahmadian A, Ehn M, Hober S. Pyrosequencing: History, biochemistry and future. Clin. Chim. Acta. 2006;363(1-2):83-94.

6. 454 Life Sciences. Bionity. 2006 [accessed 2021 Sept 31]. https://www.bionity.com/en/encyclopedia/454_Life_Sciences.html

7. Six Years After Acquisition, Roche Quietly Shutters 454. Bio-IT World. 2013 [accessed 2021 Sept 31]. https://www.bio-itworld.com/news/2013/10/16/six-years-after-acquisition-roche-quietly-shutters-454

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The very first DNA sequencing technology was created by Dr. Frederick Sanger in the 1970s who is often referred to as the ‘father of genomics’ and received a Nobel prize in 1980 for his ground-breaking invention.1 This DNA sequencing technology, called Sanger Sequencing or the Chain Termination Method, works in the following manner. 

Sanger Sequencing & Data Analysis

To prepare the target DNA for sequencing, it is first broken up into short fragments that are denatured into two single-stranded DNA (ssDNA) fragments and a shorter DNA sequence called a primer binds to the ssDNA through complementary base pairing (Figure 1A). Next, DNA polymerase (DNAP) adds deoxynucleotide triphosphates (dNTPs) in solution adjacent to the primer based on complementarity, and this process continues until it reaches the end of the ssDNA. This reaction occurs in four different reaction tubes where each also contains one of the four chain-terminating variations of the dNTPs called dideoxynucleotides (ddNTP) (Figure 1B). Whenever the ddNTPs are incorporated into the elongating strand, DNAP can no longer elongate the DNA strand, terminating the process.2 And since there are many different DNA strands being elongated and terminated in the same test tube, you end up with DNA fragments of varying lengths and molecular weights due to the incorporation of the ddNTP at each nucleotide position on the DNA fragment.2 It is important to note that these ddNTPs are labeled with radioactive molecules which is important for identification as each type of ddNTP has a unique radioactive emission.2

Once the reaction is complete, the DNA fragments are subjected to gel electrophoresis which separates the terminated DNA fragments according to size, where the shortest strands move through the gel the fastest (Figure 1C). The entire gel is then placed under an X-ray or UV light that enables visualization of the radiolabelled ddNTPs.2 Next, by moving up the gel and recording the ddNTP in the sequence, scientists can obtain the sequence of the target DNA molecule (Figure 1C). Given the very limited technology available at the time, the accuracy, robustness, and ease of use made Sanger’s Sequencing one of the most common DNA sequencing technologies used for many years to come.1,2

Figure 1: Sanger Sequencing Technology. A. The target DNA is fragmented, amplified, denatured, and bound to a primer. B. The four reaction mixtures which contain the different radiolabeled ddNTPs where the elongation and termination take place. C. Gel electrophoresis to separate the terminated fragments by size.

The Automation of Sanger Sequencing

Over the following years, Sanger Sequencing experienced several improvements that have allowed for automation of the technology, through the use of fluorescent labels called fluorophores that are bound to the chain-terminating dideoxynucleotides and computers for data acquisition and analysis.3 

Sanger Sequencing & Data Analysis 2.0

Following the same principle and upon completion of the chain-terminating reaction, all the components of this test tube are subjected to gel electrophoresis, which separates the various terminated DNA molecules according to size (Figure 2A). In this improved variation of Sanger’s Sequencing, the gel electrophoresis occurs in a capillary tube, another important aspect of automation (Figure 2B).3 Towards the end of the capillary tube, there is a laser that excites the fluorophore incorporated onto the elongating strand of the DNA molecule to generate a peak that is recorded onto a graph (Figure 2C).3 As a result, this allows scientists to obtain the entire DNA sequence once all the terminated DNA fragments have passed through the laser.

Figure 2: Automated Sanger Sequencing Technology. A. The target DNA is fragmented, amplified, denatured, and bound to a primer. B. The elongation process occurs in a single reaction mixture where the addition of fluorescently labeled ddNTP results in termination. C. Capillary gel electrophoresis to separate the terminated fragments by size and laser excitation for detection.

Sanger Sequencing provided a Launchpad for New and Innovative Technologies

Sanger Sequencing has, for the first time ever, allowed scientists to sequence DNA originating from many organisms which has improved our understanding of pathogens, the origins of disease, and the world around us. Despite what was achieved, its considerable accuracy, and widespread availability have been overshadowed by several outstanding limitations that have bottle-necked its use in modern applications.2,3 This includes how the sequence generated is derived from a single sequencing reaction, as well as the restriction that DNA fragments sequenced must be under 1000 nucleotides long, alongside the fact that the process is highly laborious and time-consuming.2 As a result, Sanger Sequencing has undergone numerous upgrades to improve and keep up to pace with the competition, which is the reason why the automated process is still used today under Thermo Fisher Scientific. However, many of the newer generations of DNA sequencing technologies have, in large part, overtaken Sanger Sequencing. Regardless, this does not discredit the fact that this first-generation DNA sequencing technology paved the way for the subsequent waves of innovative sequencing technologies that have revolutionized medicine.

References:

1. Kchouk M, Gibrat J, Elloumi M. Generations of Sequencing Technologies: From First to Next Generation. Biol. Med. 2017;09(03).

2. Sanger F, Nicklen S, Coulson A. DNA sequencing with chain-terminating inhibitors. PNAS. 1977;74(12):5463-5467.

3. Karger B, Guttman A. DNA sequencing by Capillary Electrophoresis. Electrophoresis.;30(S1):S196-S202.

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