Ever since researchers completed the Human Genome Project in 2003, a new era of genome analysis has been emerging. This has led to a more affordable, rapid and accurate method of genome analysis that is referred to as Next Generation Sequencing (NGS). This builds on the older method which researchers refer to as “first generation sequencing.”
In 1977, Fred Sanger sequenced the first entire DNA genome of a virus. Sanger went on to develop the first DNA sequencing technology that gave rise to the genome research era. He used a method called “chain termination.” This is what researchers refer to as first-generation sequencing or Sanger sequencing. It is necessary to understand Sanger sequencing in order to grasp the concepts in next-generation DNA sequencing.
The Sanger method
The Sanger method works with deoxynucleoside triphosphates (dNTPs). These base molecules bind with DNA and terminate replication because they prevent any other binding of other base molecules. The procedure begins by adding primers to a solution that contains the genes that you want to sequence. The researcher then divides the solution into four PCR reactions. Each of these reactions contains a dNTP mixture that substitutes each of the four ddNTP groups.
The next stage yields PCR results of various lengths due to random termination of replication. Researchers can then run the samples on a gel using 4 lanes and put the sequence together because it has been replicated from the same material.
To make this concept easier to understand, imagine a guessing game where you’re thinking of a number, and other people try to guess it. It’s a little bit difficult because the number is over 200 digits long. You can read the digits to yourself without the people hearing it, but you tell them where you are in the sequence. Each time someone interrupts you, you have to start over. At some point, you leave the party so that others can try to guess the 200-digit number. If they got enough clues from the interruptions, they can guess the entire number. This wouldn’t be a fun party game, but it works for gene sequencing. However, this old method is slow and expensive, so scientists invented better methods that they call Next Generation Sequencing.
Next Generation Sequencing
The latest methods of genome sequencing have benefited greatly in terms of speed and accuracy. This leads to lower staff requirements and lower costs. Greater speed is accomplished through high throughput and parallel analysis. An analogy would be comparing a first-grader who has to read a passage from a book to several PhD students who each read a passage simultaneously.
The industry has developed much more powerful and streamlined equipment since Sanger developed the original genome sequencing method. The first automatic sequencing machine was created in 1987. It used capillary electrophoresis that could perform the Sanger method without a gel. The machine, the AB370, could process 96 bases simultaneously and 500,000 bases per day. This machine was the main tool used to complete the famous human genome project.