Pyrosequencing: A Complete Overview for Researchers and Students

Table of Contents

• Introduction to Pyrosequencing
• Historical Background
• Principle of Pyrosequencing
• Process/Steps of Pyrosequencing
• Types of Pyrosequencing
• Advantages of Pyrosequencing
• Limitations of Pyrosequencing
• Applications of Pyrosequencing

Introduction to Pyrosequencing

Pyrosequencing is a sequencing technique that determines the order of nucleotides by detecting the release of pyrophosphate (PPi) as nucleotides are incorporated, allowing real-time monitoring of DNA synthesis. 

This method relies on light detection triggered by a chain reaction initiated by the release of pyrophosphate. Unlike Sanger sequencing, pyrosequencing does not use fluorescently labeled nucleotides or rely on electrophoresis for visualization. While it is particularly useful for applications requiring short reads and real-time data analysis, pyrosequencing has now largely been supplanted by newer technologies, though it played a significant role in the development of next-generation sequencing (NGS) technologies.

Historical Background

• Pyrosequencing is rooted in the pioneering work of Pal Nyren, who in 1987 demonstrated that DNA polymerization could be detected by the release of pyrophosphate.
• In 1990, Nyren collaborated with Mathias Uhlen and Bertil Pettersson to develop the solid-phase pyrosequencing method, utilizing magnetic beads and firefly luciferase for luminescence detection.
• In 1996, Mostafa Ronaghi introduced the use of the modified nucleotide deoxyadenosine α-thiotriphosphate (dATPαS) to replace the standard dATP. This modification prevented false signals in the pyrosequencing process, which typically occurred due to luciferase activity with dATP.
• In 1997, Nyren, Uhlen, Ronaghi, and Pettersson, together with Bjorn Ekstrom, founded the company Pyrosequencing AB to commercialize the technology.
• In 1998, Ronaghi introduced the enzyme apyrase to degrade unincorporated nucleotides.
• The first commercial pyrosequencing system was launched by Pyrosequencing AB in 1999.
• In 2005, the pyrosequencing technology was acquired by 454 Life Sciences, which further developed it into a high-throughput sequencing platform known as 454 Sequencing, incorporating emulsion PCR.
• In 2007, Roche acquired this technology. However, with the rise of newer sequencing technologies, the use of 454 sequencing declined, leading Roche to discontinue the production of 454 sequencers in 2013.

Principle of Pyrosequencing

The principle of pyrosequencing is rooted in the sequencing-by-synthesis approach. It involves adding nucleotides to a single-stranded DNA template and detecting the release of pyrophosphate (PPi) with each nucleotide incorporation. The released PPi triggers a series of reactions that generate a light signal, which is then used to determine the DNA sequence.

During the pyrosequencing process, DNA polymerase adds nucleotides sequentially to the template strand, releasing PPi whenever a nucleotide pairs with the complementary base. ATP sulfurylase then converts the released PPi into adenosine triphosphate (ATP). The enzyme luciferase uses this ATP to convert the substrate luciferin into oxyluciferin, resulting in light emission. This emitted light is detected by a sensor and recorded as peaks, known as a Pyrogram. The intensity of the light emitted correlates with the amount of PPi released, indicating the number of nucleotides added. Any unincorporated or excess nucleotides are degraded by the enzyme apyrase before the next nucleotide is introduced.

Process/Steps of Pyrosequencing

1. Sample Preparation

• The DNA of interest is extracted from biological samples using appropriate methods such as mechanical disruption and chemical lysis.
• The extracted DNA is then fragmented into smaller pieces, either by restriction enzymes or mechanical methods.

2. PCR Amplification

• To prepare the DNA template for sequencing, the region of interest is amplified using PCR, which generates multiple copies of each fragment.
• During amplification, a biotinylated primer is used, labeling one strand of the DNA with biotin.
• The resulting PCR product contains a biotinylated strand, which serves as the template for the sequencing reaction.
• This biotin-tagged single-stranded DNA is isolated from the PCR product using streptavidin-coated beads that bind to the biotin label.
• The template DNA is then hybridized with a sequencing primer and added to the pyrosequencing reaction.

3. Sequencing Reaction

• The necessary reagents, including the template DNA, enzymes, and substrates, are loaded into the sequencing instrument to initiate the sequencing process.
• The sequencing reaction begins with the addition of nucleotides to the template DNA.
• DNA polymerase adds complementary nucleotides to the DNA strand, releasing pyrophosphate (PPi) in the process, which triggers an enzymatic reaction cascade.

DNA template + dNTP (complementary) → DNA product + PPi + H+

• The released PPi is converted into ATP by the enzyme ATP sulfurylase in the presence of adenosine 5′ phosphosulfate (APS).

PPi + APS → ATP + SO4^2−​

• The ATP generated is used by the enzyme luciferase to convert the luciferin substrate into oxyluciferin, producing light signals that indicate the number of nucleotides added.

ATP + luciferin + O2​ → AMP + PPi + oxyluciferin + CO2 ​+ light

• The enzyme apyrase degrades ATP and any unincorporated nucleotides.
• Unincorporated nucleotide + H2O → Nucleoside + Pi
• The intensity of the emitted light is detected by sensors such as a charge-coupled device (CCD) camera and recorded as peaks.

4. Sequence Analysis

• The sequencing reaction generates pyrograms, which are graphical representations of the light signals, displaying the sequence of light peaks corresponding to the nucleotides added.
• The light signals are analyzed to determine the sequence of nucleotides.
• Multiple fragments are then assembled into a complete DNA sequence using bioinformatics tools.

Types of Pyrosequencing

a. Solid-phase pyrosequencing

Solid-phase pyrosequencing utilizes a solid substrate, such as beads, to immobilize DNA fragments for sequencing. In this method, the DNA template is immobilized on a solid surface, which serves as the template for the sequencing reaction. The sequencing process occurs on this immobilized template. Solid-phase pyrosequencing was the original pyrosequencing method developed in the late 1990s and laid the groundwork for the development of other sequencing technologies, playing a crucial role in the advancement of next-generation sequencing (NGS) methods.

b. Liquid-phase pyrosequencing

Liquid-phase pyrosequencing conducts sequencing in a solution. Although less commonly used in high-throughput applications, this method simplifies the process and reduces some of the complexities associated with solid-phase techniques. In liquid-phase pyrosequencing, single-stranded DNA fragments are prepared and hybridized to a sequencing primer within a liquid solution. Nucleotides are added sequentially in the liquid phase, and each nucleotide incorporation releases pyrophosphate, which is then converted to ATP and detected as a light signal.

c. 454 pyrosequencing

454 pyrosequencing was the first commercially successful NGS technology, developed by 454 Life Sciences, which integrates emulsion PCR with pyrosequencing. In this method, DNA fragments are amplified within tiny droplets of an oil-water emulsion. The process involves isolating and fragmenting DNA, amplifying these fragments on beads within the emulsion, and performing sequencing by synthesis in a picotiter plate. Each bead within the emulsion contains a single DNA template.

d. Microfluidic pyrosequencing

Microfluidic pyrosequencing combines pyrosequencing technology with microfluidics. This method involves trapping DNA on microbeads within a filter chamber, where DNA is prepared and introduced into microfluidic channels. Amplification and sequencing take place within these channels. Microfluidic pyrosequencing addresses some limitations of traditional microtiter plates, such as high reagent costs and dilution effects, which limit throughput and cost-efficiency for large-scale samples. Variations of microfluidic pyrosequencing include methods that use capillaries to deliver nucleotides into a microchamber and digital microfluidics that utilize electrically controlled droplets for sequencing.

Advantages of Pyrosequencing

• Pyrosequencing offers real-time sequencing data, allowing for continuous monitoring of the sequencing process as it happens.
• This method is high-throughput, capable of processing multiple samples simultaneously, making it valuable for large-scale genome sequencing projects.
• The results of pyrosequencing are directly converted into Pyrogram peaks, eliminating the need for post-sequencing electrophoresis, thereby reducing both time and costs.
• Pyrosequencing requires fewer sample preparation and pre-processing steps compared to other sequencing methods.
• Unlike other techniques, pyrosequencing does not require fluorolabeling of nucleotides. The use of natural nucleotides not only reduces costs but also simplifies the overall process.

Limitations of Pyrosequencing

• Pyrosequencing generates shorter read lengths compared to other sequencing methods, which can restrict its application in sequencing large genomes and repetitive sequences.
• The method struggles with accurately detecting long homopolymer regions or sequences with repeated nucleotides, often leading to sequencing errors.
• While pyrosequencing is less expensive than some next-generation sequencing methods, the cost per base pair is higher compared to other high-throughput techniques.
• Due to advancements in sequencing technologies that offer longer read lengths and higher throughput at lower costs, pyrosequencing has largely been supplanted by more advanced methods.

Applications of Pyrosequencing

• Pyrosequencing is employed to identify and study genetic variations, such as single nucleotide polymorphisms (SNPs) and other mutations, which are valuable in genetic mapping. This technique is instrumental in understanding mutations associated with various diseases and is useful in genetic screening and personalized medicine.
• It is also utilized to study DNA methylation patterns, which provide insights into gene regulation and the epigenetic changes linked to different diseases.
• In cancer research, pyrosequencing is used to identify tumor-specific mutations, aiding in the discovery of biomarkers and the development of targeted therapies.
• The method is applied in microbial identification, which is particularly useful in metagenomics and microbiome studies.
• Additionally, pyrosequencing can be used to identify various pathogens, making it valuable in clinical diagnostics and epidemiological studies.