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
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.
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
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.
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