What is the Difference Between Sanger Sequencing and Pyrosequencing

The main difference between Sanger sequencing and pyrosequencing is that Sanger sequencing is a DNA sequencing approach that uses the dideoxy chain termination method, whereas pyrosequencing is a DNA sequencing approach based on the sequencing-by-synthesis principle. Therefore, in Sanger sequencing, the identification of nucleotides is by capillary electrophoresis after the amplification of whole DNA fragment while in pyrosequencing, the identification of nucleotides is done with the release of pyrophosphate during the synthesis.  

Sanger sequencing and pyrosequencing are two methods of DNA sequencing; former is the ‘gold standard’ for most targets while the latter is the first alternative to the conventional Sanger sequencing method. 

Key Areas Covered 

1. What is Sanger Sequencing
     – Definition, Process, Importance
2. What is Pyrosequencing
     – Definition, Process, Importance
3. What are the Similarities Between Sanger Sequencing and Pyrosequencing
      – Outline of Common Features
4. What is the Difference Between Sanger Sequencing and Pyrosequencing
      – Comparison of Key Differences

Key Terms 

DNA Sequencing, PCR, Pyrophosphate, Pyrosequencing, Sanger Sequencing, Sensitivity

What is Sanger Sequencing 

Sanger sequencing is the first generation method of DNA sequencing first developed by Fredric Sanger in 1977. Moreover, the basis of Sanger sequencing is the dideoxy chain termination method. 

Sanger Sequencing – Procedure 

In Sanger sequencing, DNA polymerase is responsible for the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) during in vitro DNA synthesis. Therefore, the dideoxynucleotides (ddNTPs) are fluorescent-labelled into the amplicon by PCR. Here, the ddATP is labelled with green dye; the ddGTP is labelled with yellow dye; the ddCTP is labelled with blue, and the ddTTP is labelled with red dye) Then, the resulting amplicons are separated by capillary electrophoresis while detecting the fluorescent-labelled nucleotides.

Figure 1: Sanger Sequencing Method

Sanger Sequencing – Importance 

However, Sanger sequencing method has several limitations including the incapability to process longer sequencing output, parallel analysis of fewer samples, the inability of the total automation of sample preparation, higher cost, sequencing errors, less sensitivity (10-20%), which is insufficient for the detection of low-level mutant alleles, etc. Despite these limitations, it is the ‘gold standard’ for sequencing in many clinical procedures. 

What is Pyrosequencing 

Pyrosequencing is the first alternative to conventional Sanger sequencing. It is a type of next-generation sequencing developed at the Royal Institute of Technology (KTH). Moreover, this method is based on the luminometric detection of pyrophosphate (PPi) released during primer-directed DNA polymerase-catalyzed nucleotide incorporation.  

Pyrosequencing – Procedure 

Generally, four enzymes are used in this method to accurately detect the incorporated nucleotides. They are DNA polymerase, ATP sulfurylase, luciferase, and apyrase. Moreover, the sequencing primer hybridizes to a single-stranded DNA biotin-labelled template. Additionally, the four deoxynucleotide triphosphates (dNTPs), adenosine 5’ phosphosulfate (APS) and luciferin are the substrates in the reaction mixture. 

Figure 2: Pyrosequencing Method

When the polymerization cascade starts, inorganic PPi releases as a result of nucleotide incorporation by the polymerase. However, the quantity of the released PPi is equimolar to the amount of incorporated nucleotide in each cycle. Subsequently, ATP sulfurylase converts the released PPi to ATP in the presence of APS in a quantitative manner. The generated ATP drives the conversion of luciferin to oxyluciferin mediated by the luciferase enzyme. Also, this reaction proportionally generates visible light to the amount of ATPs. Then, this light can be detected at the 560 nm wavelength.

Moreover, the main function of the apyrase enzyme is to continuously degrade ATP as well as non-incorporated dNTPs in the reaction mixture. Therefore, new dNTPs have to be added to the reaction one at a time in a certain time interval, which is 65 s. As the added nucleotide is known, the sequence of the template can be determined. 

Pyrosequencing – Importance 

Moreover, pyrosequencing is a widely applicable technique with high accuracy, parallel processing, and easily automated. Also, it avoids the usage of labeled primers, labeled nucleotides, and gel electrophoresis. Additionally, it is suitable for both confirmatory sequencing and de novo sequencing. Further, the main important feature of pyrosequencing is its sequencing depth, which allows the detection of variants with high sensitivity. However, the main drawback of the technique is its suitability to sequence up to several hundreds of bases. 

Similarities Between Sanger Sequencing and Pyrosequencing 

• Sanger sequencing and pyrosequencing are two approaches to DNA sequencing. 
• They are responsible for the identification of the nucleotide sequence of a DNA fragment of interest. 
• Both are better for sequencing smaller DNA fragments. 
• However, they have their own applications depending on their sequencing procedure and benefits. 

Difference Between Sanger Sequencing and Pyrosequencing 

Definition 

Sanger sequencing refers to a method of DNA sequencing by the selective incorporation of chain-terminating dideoxynucleotides, while pyrosequencing refers to a method of DNA sequencing based on the sequencing-by-synthesis principle. 

Type of Sequencing 

Sanger sequencing is the first generation sequencing approach, while pyrosequencing is next-generation sequencing chemistry, which is a second-generation sequencing approach. 

Correlation 

Moreover, Sanger sequencing is the conventional method and the ‘gold standard’ for most targets, while pyrosequencing is the first alternative to the conventional sequencing method. 

Invention 

Frederick Sanger and colleagues were the first to develop Sanger sequencing in 1977 while Pål Nyrén and his student Mostafa Ronaghi were the first to develop pyrosequencing, at the Royal Institute of Technology in Stockholm in 1996. 

Commercialization 

While Sanger sequencing is first commercialized by Applied Biosystems, pyrosequencing is used in Roche 454 and GS FLX Titanium platforms. 

Principle 

Above all, the main difference between Sanger sequencing and pyrosequencing is that Sanger sequencing uses the dideoxy chain termination method, while pyrosequencing is based on the sequencing-by-synthesis principle.  

Identification of Nucleotides 

In Sanger sequencing, the identification of nucleotides is by capillary electrophoresis after the amplification of the whole DNA fragment while in pyrosequencing, the identification of nucleotides is done with the release of pyrophosphate during the synthesis.  

Detection 

Furthermore, Sanger sequencing involves the detection of fluorescent light, while pyrosequencing involves the detection of visible light at 560 nm. 

Length of DNA Fragments 

Additionally, Sanger sequencing can read up to 800 to 1000 base pairs while pyrosequencing can read up to 300-500 base pairs. 

Significance 

Sanger sequencing is a complex process with many steps, while pyrosequencing is a less complex process with fewer steps. 

Sensitivity 

Also, another difference between Sanger sequencing and pyrosequencing is that Sanger sequencing has a lower sensitivity, while pyrosequencing has a higher sensitivity. 

Conclusion 

Sanger sequencing is the first generation sequencing approach, which is the conventional method of sequencing. Also, it is the ‘gold standard’ for many targets. However, it uses the dideoxy chain termination method followed by capillary electrophoresis. On the other hand, pyrosequencing is the first alternative to Sanger sequencing, and it is a type of next-generation sequencing. Further, it has a higher sensitivity and fewer steps to cover. Generally, it uses the sequencing-by-synthesis method, which determines nucleotides during the synthesis of DNA fragment as it is. Therefore, the main difference between Sanger sequencing and pyrosequencing is the method of sequencing and their benefits. 

References:

1. Fakruddin, Md, and Abhijit Chowdhury. “Pyrosequencing-An Alternative to Traditional Sanger Sequencing.” American Journal of Biochemistry and Biotechnology, vol. 8, no. 1, 2012, pp. 14–20., doi:10.3844/ajbbsp.2012.14.20.

Image Courtesy:

1. “Sanger-sequencing” By Estevezj – Own work (CC BY-SA 3.0) via Commons Wikimedia   
2. “How Pyrosequencing Works” By “Jacopo Pompilii, DensityDesign Research Lab”. – Own work (CC BY-SA 4.0)via Commons Wikimedia