What is Pyrophosphate Sequencing (Pyrosequencing)?

Pyrophosphate sequencing represents an innovative enzymatic cascade chemiluminescence sequencing method driven by four key enzymes: DNA polymerase, ATP sulfurylase, luciferase, and adenosine triphosphate bisphosphatase (apyrase). This cutting-edge technology enables real-time detection of biological light signals emitted during DNA synthesis, setting a groundbreaking precedent for simultaneous sequencing and synthesis.

In the realm of early tumor screening and diagnosis, numerous methodologies focus on epigenetic methylation detection. Pyrophosphate sequencing stands out as the "gold standard" for methylation verification. Today, let’s delve deeper into the principles and applications of pyrophosphate sequencing technology to enhance our understanding of its significance.

Pyrophosphate Sequencing Involves Key Components

Pyrophosphate sequencing comprises template DNA, dNTPs (dATP, dGTP, dTTP, dCTP), enzyme mixtures, and substrates.

The enzyme mixture consists of four enzymes: DNA polymerase, ATP sulfurylase, luciferase, and adenosine triphosphate bisphosphatase (apyrase).

The substrates encompass two essential substances: 5′-phosphonosulfate (APS) and luciferin.

Workflow of Pyrophosphate Sequencing

Pyrosequencing represents an innovative enzyme cascade chemiluminescent sequencing technology driven by the catalytic actions of DNA polymerase, ATP sulfurylase, luciferase, and adenosine triphosphate bisphosphatase. This groundbreaking approach pioneer sequencing-while-synthesizing, offering real-time detection of subtle bio-optical signals during the DNA contraction reaction.

• In each round of the sequencing reaction, only one deoxynucleotide triphosphate (dNTP) is introduced. If the dNTP pairs with the template, DNA polymerase adds it to the 3′ end of the sequencing primer, releasing an equal amount of pyrophosphate (PPi).
• ATP sulfurylase facilitates the combination of released PPi with 5′-phosphonosulfate (APS), forming ATP. Subsequently, luciferase catalyzes the reaction between ATP and luciferin, producing oxidized luciferin (oxyluciferin) and emitting visible light. The CCD sensor captures this light signal, converting it into detection peaks. The height of each peak is directly proportional to the incorporated nucleotides.
• Adenosine triphosphate bisphosphatase (apyrase) continually degrades unincorporated nucleotides and ATP, extinguishing the light signal and regenerating the reaction system to yield the complete result.
• Repeat the process by adding another dNTP and cycling. This iterative cycle allows real-time reading of the exact nucleotide sequence of the template DNA based on the added dNTP type and the fluorescence signal intensity (peak level).

Interpretation of Sequencing Results

The generated light intensity serves as an indicator of the successful pairing of specific deoxynucleotide triphosphates (dNTPs) with the template, reflecting their incorporation into the newly synthesized chain. The height of the peaks corresponds directly to the number of dNTPs successfully integrated into the emerging chain.

The absence of a peak signifies the failure of dNTP incorporation, while a peak at 1x height denotes successful doping with one dNTP. Peaks at 2x and 3x heights represent the successful doping of two and three dNTPs, respectively. Following the interpretation of the peak diagram and the principles of base complementary pairing, the template DNA sequence can be determined.

Pyrosequencing Platform

The 454 sequencing technology represents an ultra-high-throughput genome sequencing system grounded in pyrophosphate sequencing. In this method, a specialized plate known as the "Pico TiterPlate" (PTP) is employed, featuring over 1.6 million wells composed of optical fibers containing diverse enzymes and substrates for chemiluminescent reactions. The sequencing process initiates with the cycling of four bases—T, A, C, and G—from separate reagent bottles into the PTP plate, one base at a time.

Upon base pairing, the release of pyrophosphate occurs. This pyrophosphate undergoes a series of reactions, including synthesis and chemiluminescence, ultimately transforming fluorescein into oxidized fluorescein and emitting a light signal. The highly sensitive CCD integrated into the instrument captures this real-time light signal. Each paired base with the sequencing template corresponds to the capture of a light signal, enabling precise and rapid determination of the base sequence in the tested template.

Pyrosequencing vs. Sanger Sequencing vs. Illumina Sequencing

Sanger Sequencing, as the pioneering DNA sequencing technology of its generation, boasts a remarkable sequencing read length of up to 1000 bp and an impressive accuracy level of 99.999%. However, its widespread application has been hindered by high sequencing costs and limited throughput.

Please read our blog: Sanger Sequencing: Introduction, Principle, and Protocol.

The 454 high-throughput sequencing system, leveraging pyrophosphate sequencing, addresses some of these limitations with notable advantages in read length. This feature enhances the efficiency and accuracy of subsequent splicing work, making it an excellent choice for applications like whole-genome sequencing, transcriptome analysis, and genome structure analysis. Despite its strengths, the use of pyrophosphate sequencing introduces challenges, such as limitations in throughput due to the detection of transient luminescence and reduced accuracy in detecting homodimers. The longer the homodimer, the greater the potential for errors. Additionally, the sequencing cost is relatively high compared to alternative high-throughput platforms.

Following the success of 454 sequencing, a wave of new parallel sequencing technologies emerged. Illumina sequencing platforms, utilizing synthesis-as-you-sequence technology, have become the primary commercially available high-throughput parallel sequencing option. Next-generation sequencing (NGS) platforms, led by Illumina, have become dominant, offering high-throughput sequencing at a more affordable cost. However, these platforms are typically constrained in read length, typically generating reads between 50 and 500 base pairs (bp).

The Illumina HiSeq X System stands out with its capacity for whole-genome sequencing beyond human species. It is versatile, catering to applications such as whole exome sequencing, transcriptome sequencing, single-cell analysis, and multi-omics studies.

Application of Pyrophosphate Sequencing

Pyrophosphate sequencing technology stands out by eliminating the need for specialized fluorescent labeling and electrophoresis. Its user-friendly operation, coupled with sequencing reproducibility and accuracy comparable to Sanger sequencing, distinguishes it as a rapid and efficient alternative—boasting sequencing speeds 100 times faster than traditional methods. However, the reliance on transient luminescence does impose some limitations on throughput, and accuracy in detecting homopolymers diminishes with their length, with longer homopolymers presenting a higher risk of errors.

Pyrophosphate sequencing technology finds diverse applications across multiple fields, including single nucleotide polymorphisms (SNP), insertions/deletions (indels), short tandem repeats, human leukocyte antigen (HLA) typing, gene copy number analysis, RNA allelic imbalance, and methylation analysis. Notably, it excels in the rapid sequencing of known short DNA sequences (20-50bp).

As a cutting-edge sequence analysis technology, pyrosequencing emerges as the gold standard for methylation detection. Its ability to swiftly determine methylation frequency, qualitatively and quantitatively analyze methylation sites in samples, positions it at the forefront of methylation detection methodologies.