30 May 2023

Understanding Next Generation Sequencing Methods

Next generation sequencing (NGS) is innovative technology helping scientists unlock the DNA puzzle. It is an approach that opens doors to personalized medical care that is both predictive and preventive. 

When people hear the term next generation sequencing, they likely think of second-generation (2G) technologies. As the importance of this revolutionary bioscience became clear, the technology evolved. Today, there are third- and fourth-generation technologies involved in NGS that move past the initial study to focus on more advanced principles. 

What is Next Generation Sequencing? 

NGS allows researchers to understand the intricate puzzle of DNA. DNA is a code that controls the structure of proteins and the function of the cells that make up the body of the organism.

The importance of DNA goes beyond just physical characteristics. DNA plays a role in disease processes, as well. For example, inherited forms of breast cancer are directly associated with two gene mutations. 

NGS allows researchers to read the order of the chemical bases in DNA. They can see what code each gene holds and understand it. 

Being able to decode DNA allows medical researchers to understand disease processes better and develop ways to change the code and create cures that were inconceivable 50 years ago. Today, labs use a number of methods to investigate DNA sequences. 

Second-Generation Sequencing

Second-generation sequencing is the most established technology. Several methods fit into this category, and we can break them down into subdivisions, such as:

  • Sequencing by ligation
  • Sequencing by synthesis
  • Proton detection
  • Pyrosequencing
  • Reversible terminator

This breakdown of 2G technologies focuses on the underlying detection chemistries. For example, sequencing by ligation utilizes an enzyme called DNA ligase to pinpoint nucleotides present in a key position in the DNA. On the other hand, sequencing by synthesis uses fluorescently-labeled nucleotides to sequence the chemical bases. Proton detection counts the hydrogen ions as the DNA is polymerized. 

2G sequencing allows researchers to get results quickly and for less cost. 

Third-Generation Sequencing

2G sequencing requires polymerase chain reaction (PCR) to amplify the material enough to sequence it. Third-generation sequencing (3G) eliminates that need.

3G technology is able to sequence single molecules and does not require amplification. Initially developed by scientists in the Department of Applied Physics at the California Institute of Technology in 2003, 3G uses fluorescently labeled nucleotides incorporated into the DNA strands. This provides longer reads than 2G, but it can come with problems. Studies indicate that 3G processing can lead to higher error rates. It also costs more. 

Fourth-Generation Sequencing

Fourth-generation sequencing (4G) utilizes the same single-molecule sequencing but adds nanopore technology for real-time analysis. This sequencing uses tiny biopores called nanopores due to their size. They allow one single molecule to pass through the membrane at a time. As each molecule comes through, the technology identifies it. 

Although fourth-generation sequencing combines traditional methods with state-of-the-art technology, the process is costly. It does give scientists the fastest route to sequencing whole genomes, but the error rate is still high. 

One thing is clear — next generation sequencing has a crucial role to play in the future of medicine. As technology evolves, the process will become more accurate and affordable.