NGS (Next Generation Sequencing)

NGS revolutionized the world of genomics in 2007, introducing a completely new way of DNA sequencing and drastically reducing the cost and time required for genome sequencing. An example of the importance of this method is that the Human Genome Project took 10 years and cost more than 3 billion dollars; today, however, the new generation of sequencing can sequence the entire human genome in less than 2,000 dollars in one day. One of the reasons for this is that samples are prepared faster than previous techniques; for example, there is no need to produce DNA libraries for bacteria anymore. In addition to saving cost and time, the number of sequenced genomes has increased significantly since the discovery of these techniques.
The Illumina sequencing technique is based on the dideoxy technique, but it has benefited from many other innovations. During this process, nucleotides are attached to a detachable fluorescent molecule, each of which has a different color, as well as a chain terminating chemical. Instead of the ‘3-OH’ group that terminates the sequence in normal deoxy sequencing, in this technique the nucleotides have a chemical group that can block ‘3-OH’ and prevent further replication by DNA polymerase. These groups can be isolated enzymatically. As with other new generation sequencing technologies, the first step in this technology is to prepare DNA for amplification by PCR. In fact, instead of using PCR products or cloned sequences, these techniques start with genomic DNA. The extracted DNA is broken into smaller pieces that are about 200 to 600 bp in length. Short sequences called “Adapters” attach to DNA fragments. The DNA molecules are then denatured to become single stranded. After these preparation steps, the DNA fragments are attached to the surface of a flow cell, and then the surface of the flow cell is rinsed so that the unbound DNAs do not remain on the surface. The flow cell surface has primers that complement the surface-mounted adapters. Adapters and primers must be far enough apart that the detector on the floor of the flow cell detects them in separate positions. Similar to 454 sequencing technique, DNA fragments fixed at the flow cell surface are replicated by incubation with DNA polymerase and deoxynucleotides in a process called bridge sequencing. The primers required to amplify DNA fragments are attached to the flow cell; As a result, DNA binds to the primer to form a bridge-like form. These fragments are duplicated, released and then, denatured to form a mass of similar DNA fragments.

In the Illumina technique, DNA is prepared for the amplification process by extracting and dividing the sample into smaller pieces. The adapters attach to the ends of the components and then to the complementary oligonucleotides at the flow cell surface. The parts are connected in such a way that their distance from each other can be detected by the detector device on the floor of the flow cell. Each sample molecule is amplified and then denatured to become single stranded. After connecting the primer to one of the ends of the sample strings and determining the sequence base-to-base during the synthesis of the new string, the sequence is created (Refer to the original text for more details).
The sequencing process is then performed in the following order: Four nucleotides labeled with fluorescent dyes, along with the DNA polymerase enzyme, are added to the billions of DNA strands fixed on a slide. In each mass, only nucleotides complementing the sample sequence are covalently attached and unbound nucleotides are eluted. The high-resolution digital camera then captures an image that identifies the nucleotides added to the chain in each mass, after the laser is emitted and the fluorescent dyes are activated. The fluorescent label and the 3-OH blocking group are enzymatically separated and the process is repeated several times. In this way, billions of sequencing reactions take place simultaneously. By tracking color changes in each mass, the DNA sequence at each spot is determined. Although each sequence alone is relatively short (about 200 nucleotides), billions of them are performed simultaneously to sequence several human genomes in one day. Base-to-base sequence analysis has resulted in high accuracy of this method.

According to Illumina sequencing, this reaction takes place simultaneously in billions of DNA blocks. This method uses a digital color camera that quickly scans all DNA blocks at each time the nucleotide binds. The sequence of each mass is then determined from the pattern of color changes that occurs as the replication reaction progresses step by step. Each cycle of altered nucleotide fusion and separation of the 3-OH blocker and the fluorescent molecule takes less than an hour. Each mass in the slide also contains many copies of different random fragments in the genome. In the mass preparation phase, a specific DNA sequence determined by the researcher is added to each copy in each mass, and a primer that complements this sequence is used to initiate the DNA polymerase replication process.

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