Principle, Steps, Reactions, Types, Uses microbiologystudy

Pyrosequencing is a sequencing method that determines the sequence of nucleotides by detecting the release of pyrophosphate (PP_i) during nucleotide incorporation and monitors DNA synthesis in real-time.

Pyrosequencing relies on light detection which occurs due to a chain reaction initiated by the release of pyrophosphate. Unlike Sanger sequencing, this method does not use fluorescently labeled nucleotides and does not rely on electrophoresis for visualization. Pyrosequencing is useful for applications that require short reads and real-time data analysis. This method has now been largely replaced by several new technologies but it laid the groundwork for the development of next-generation sequencing (NGS) technologies.

Historical Background

• Pyrosequencing is based on the foundational work of Pal Nyren, who demonstrated in 1987 that DNA polymerization can be detected by the release of pyrophosphate. 
• In 1990, he worked together with Mathias Uhlen and Bertil Pettersson to develop the solid-phase pyrosequencing method where they used magnetic beads and firefly luciferase for luminescence detection. 
• In 1996, Mostafa Ronaghi introduced the use of the modified nucleotide deoxyadenosine α-thiotriphosphate (dATPαS) instead of using the normal dATP. This prevented false signals in the pyrosequencing process that usually occurred due to the activity of luciferase with dATP. 
• In 1997, Nyren, Uhlen, Ronaghi, and Pettersson along with Bjorn Ekstrom founded the company Pyrosequencing AB to commercialize this technology. 
• In 1998, Ronaghi introduced the enzyme apyrase to degrade unincorporated nucleotides.
• The first commercially available pyrosequencing system was launched by Pyrosequencing AB in 1999. 
• In 2005, the pyrosequencing technology was acquired by 454 Life Sciences who further developed it into a high-throughput sequencing platform called 454 Sequencing which used emulsion PCR.
• In 2007, this technology was acquired by Roche. Due to the advancements of newer sequencing technologies, the use of 454 sequencing declined and Roche decided to discontinue the production of 454 sequencers in 2013.

Principle of Pyrosequencing

The principle of pyrosequencing is based on the sequencing-by-synthesis method. It is based on the addition of nucleotides to a single-stranded DNA template and the detection of the release of pyrophosphate as each nucleotide is added. The released PP_i initiates a series of reactions that produce a light signal which is used to determine the DNA sequence.

In the pyrosequencing process, DNA polymerase adds nucleotides sequentially to the template strand and releases PP_i when the nucleotide matches the complementary base. ATP sulfurylase converts the released PP_i into adenosine triphosphate (ATP). Then the enzyme luciferase uses the ATP to convert the substrate luciferin into oxyluciferin which emits light as a result. The light emitted is detected by a sensor and recorded as peaks called Pyrogram. The intensity of the emitted light correlates with the amount of PPi released and thus reflects the number of nucleotides added. Any unincorporated or excess nucleotides are broken down by the enzyme apyrase before the next nucleotide is added.

Process/Steps of Pyrosequencing

1. Sample Preparation

• DNA of interest is extracted from biological samples using different suitable methods like mechanical disruption and chemical lysis.
• The extracted DNA is fragmented into small pieces with restriction enzymes or by using mechanical methods.

2. PCR Amplification

• The DNA template for sequencing is prepared by amplifying the region of interest using PCR which generates multiple copies of each fragment. 
• The fragmented DNA is amplified using a biotinylated primer which labels one strand with biotin. 
• The PCR product contains one biotinylated strand that is used 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 hybridized with a sequencing primer and then 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 reaction.
• The sequencing reaction begins with the addition of nucleotides to the template DNA. 
• DNA polymerase adds the complementary nucleotide into the DNA strand. This releases pyrophosphate which initiates an enzymatic reaction cascade.
  DNA template + dNTP (complementary) → DNA product + PP_i + H⁺
• The released PP_i is converted into ATP by the enzyme ATP sulfurylase in the presence of the substrate adenosine 5′ phosphosulfate (APS).
  PP_i + APS → ATP + SO₄²⁻​
• The ATP generated is used to convert the luciferin substrate to oxyluciferin by the enzyme luciferase. This produces light signals that indicate how many nucleotides were added.
  ATP + luciferin + O₂​ → AMP + PP_i + oxyluciferin + CO₂ ​+ light
• The enzyme apyrase degrades ATP and any unused or unincorporated nucleotides.
  Unincorporated nucleotide + H2O → Nucleoside + Pi
• The intensity of the emitted light is detected with sensors like a charge-coupled device (CCD) camera and recorded as peaks.

4. Sequence Analysis

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

Pyrosequencing Video Animation

Types of Pyrosequencing

a. Solid-phase pyrosequencing

Solid-phase pyrosequencing uses a solid substrate such as beads to immobilize DNA fragments for sequencing. This method involves immobilizing the DNA template on a solid surface which is then used as a template for sequencing reaction. The sequencing reaction occurs on this immobilized template. Solid-phase pyrosequencing is the original method of pyrosequencing that was developed in the late 1990s. This method provided the foundation for the development of other sequencing technologies and played a significant role in the development of NGS methods.

b. Liquid-phase pyrosequencing

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

c. 454 pyrosequencing

454 pyrosequencing is the first successfully commercialized NGS technology developed by 454 Life Sciences that combines 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 oil-water emulsions, and sequencing by synthesis in a picotiter plate. Each bead in the emulsion contains a single template. 

d. Microfluidic pyrosequencing

Microfluidic pyrosequencing combines pyrosequencing technology with microfluidics. This method traps DNA on microbeads within a filter chamber. DNA is prepared and introduced into microfluidic channels. Amplification and sequencing occur within these channels. This microfluidic method overcomes some of the limitations of traditional microtiter plates, such as high reagent costs and dilution effects. The conventional 96-microtiter plate format limits the throughput and cost-efficiency for large-scale samples. Besides the flow-through microfluidic device, there are other variations of microfluidic pyrosequencing. Some methods use capillaries to deliver nucleotides into a microchamber. Other methods include digital microfluidics that use electrically controlled droplets to perform sequencing.

Advantages of Pyrosequencing

• Pyrosequencing provides real-time sequencing data that allows monitoring of the sequencing process as it occurs.
• It is a high-throughput method that can process multiple samples simultaneously which is useful in large-scale genome sequencing.
• Pyrosequencing results are directly converted into Pyrogram peaks. This removes the need for post-sequencing electrophoresis for analysis which reduces time and cost.
• Pyrosequencing involves fewer sample preparation steps and pre-processing steps compared to other sequencing methods.
• Pyrosequencing does not require fluorolabeling of nucleotides. The use of natural nucleotides reduces costs and simplifies the process.

Limitations of Pyrosequencing

• Pyrosequencing produces shorter read lengths compared to other sequencing methods. This can limit its use for sequencing large genomes and repetitive sequences.
• Pyrosequencing cannot accurately detect long homopolymers or sequences that contain repeated nucleotides which leads to sequencing errors.
• Although pyrosequencing is less expensive than some next-generation sequencing methods, the cost per base pair is higher compared to other high-throughput methods. 
• Pyrosequencing has been largely replaced by more advanced sequencing technologies that offer longer read lengths and higher throughput at a lower cost. 

Applications of Pyrosequencing

• Pyrosequencing is used to identify and study genetic variations such as single nucleotide polymorphisms (SNPs) and other mutations which is useful in genetic mapping. This also helps us to understand the mutations associated with different diseases and is useful in genetic screening and personalized medicine.
• Pyrosequencing is also used to study DNA methylation patterns which help us understand gene regulation and epigenetic changes associated with different diseases.
• Pyrosequencing has applications in cancer research where it is used to identify tumor-specific mutations. This is useful to discover biomarkers and to develop targeted therapies.
• Pyrosequencing is used in microbial identification useful in metagenomics and microbiome studies.  
• Pyrosequencing can be used to identify different pathogens, making it useful in clinical diagnostics and epidemiological studies.

References

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