Next Generation Sequencing and Sanger: What are the differences?

In this article, we will explore what NGS is and how it compares to the traditional Sanger method, as well as its applications and the different types of technology currently available.

In this article, we will explore what NGS is and how it compares to the traditional Sanger method, as well as its applications and the different types of technology currently available. 

  

What is NGS? 

  

Next Generation Sequencing (NGS) is an umbrella term applied to a set of modern techniques that allow the obtention of nucleotide sequences of DNA and RNA molecules from various organisms, such as humans, fungi, animals, plants and viruses. 

  

It is possible to obtain the nucleotide sequences of a range of different biological samples including blood, saliva, milk, tissue from human biopsies, plant roots, leaves and other organs and tissues. 

  

How is NGS applied? 

  

In identifying pathogenic mutations in various human sequences, quantifying the impact of a combination of variants that result in complex characteristics, or even studying the adaptive history of human beings, to answer small and big questions. 

 

As an example, in the medical context there’s the diagnosis of genetic diseases – such as in the identification of rare diseases and in cancer research – which often have hereditary aspects and whose mutational characterization is of utmost importance. 

  

Several other applications are possible: there’s the context of biodiversity or in microbiome studies – for the identification of microbial genera and species present in samples of soil, water, human and animal feces and others; and in the analysis of evolution and ecology for the understanding of variability in populations at different geographic and temporal scales. 

  

Here we will explore the basis of sequencing technologies and in future posts you will discover even more about these applications. 

  

Is Sanger sequencing NGS? 

  

No, the sequencing volume obtained through NGS is much larger, allowing the sequencing of millions of fragments simultaneously, while in the Sanger method, even more robust protocols generate small volumes of data. 

  

Sanger 

  

Sanger sequencing allows the determination of the nucleotide sequence in a DNA fragment. This method, which is based on the termination of synthesis in the nucleotide chain uses a DNA template strand of interest, a DNA polymerase enzyme, conventional nucleotides and modified nucleotides (di-deoxynucleotides). DNA polymerase performs its work by incorporating nucleotides into the template strand – however, its work is made impossible after the addition of a di-deoxynucleotide, which prevents the elongation of the newly created strand. This generates DNA fragments of different sizes based on the original template strand. 

  

In the classical method, four reactions like the described above were necessary, one for each modified and radioactively labeled nucleotide. Subsequently, fluorescent labeling of each type of nucleotide allowed this chain elongation and termination step to be performed with just one reaction. After the fragments are generated, they were then separated by electrophoresis according to their sizes, and the nucleotide sequence can then be deciphered. 

  

NGS 

  

Next-generation sequencing (NGS) uses methodologies that are different from the Sanger method, although some technologies have similarities, such as the addition of fluorescent nucleotides to a growing template strand by a DNA polymerase. When NGS sequencing emerged, the focus was on developing protocols that would speed up data generation and lower the cost of sequencing – while maintaining or improving the quality of the results obtained. In next-generation sequencing, there is massive parallel processing of DNA fragments, which allows a greater amount of data to be generated more quickly. NGS sequencing offers better cost-effectiveness, higher resolution and sensitivity, and greater multiplexing capacity (processing multiple samples simultaneously) with barcoding technologies (labeling, barcoding). 

References 

Goodwin, Sara, John D. McPherson, and W. Richard McCombie. “Coming of age: ten years of next-generation sequencing technologies.” Nature reviews genetics 17.6 (2016): 333-351. 

Ashley, Euan A. “Towards precision medicine.” Nature Reviews Genetics 17.9 (2016): 507-522. 

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