The completion of the Human Genome Project in 2003 ushered in a new era of rapid, affordable, and accurate genome analysis—called Next Generation Sequencing (NGS). NGS builds upon “first generation sequencing” technologies to yield accurate and cost-effective sequencing results. Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology and using NGS an entire human genome can be sequenced within a single day.
The technologies used for NGS allow sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such revolutionized the study of genomics and molecular biology. The biggest advances in genome sequencing have been increasing speed and accuracy, resulting in reduction in manpower and cost which enhanced the research in genomics.
- Fluorescently labeled chain-terminating ddNTPs are added to the PCR reaction mix.
- By the Sanger sequencing method, PCR products of various lengths are created, and then separated according to their size.
- Size is measured by the PCR product’s overall negative charge. The more negative the charge, the longer the fragment.
- Take an example of ddNTP incorporation and imagine that the bold letters are ddNTPs with a fluorochrome attached. As the fragments are pulled toward the positive electrode of a capillary, they pass a laser beam that triggers a flash of light from the fluorochrome attached to the ddNTP that is characteristic of the base type (for example, green for A, yellow for T, blue for G, red for C).
- Thus, the genome is carefully read and analyzed to study the genome accurately!
Techniques for Next Generation Sequencing:
Pyrosequencing is based on the ‘sequencing by synthesis’ principle, where a complementary strand is synthesized in the presence of polymerase enzyme. In contrast to using dideoxynucleotides to terminate chain amplification (as in Sanger sequencing), pyrosequencing instead detects the release of pyrophosphate when nucleotides are added to the DNA chain.
Sequencing by ligation (SOLiD):
SOLiD is an enzymatic method of sequencing that uses DNA ligase, an enzyme used widely in biotechnology for its ability to ligate double-stranded DNA strands. Emulsion PCR is used to immobilise/amplify a ssDNA primer-binding region (known as an adapter) which has been conjugated to the target sequence (i.e. the sequence that is to be sequenced) on a bead.
- By understanding the sequence of DNA, researchers have been able to elucidate the structure and function of proteins as well as RNA and have gained an understanding of the underlying causes of disease.
- Next Generation Sequencing (NGS) is a powerful platform that has enabled the sequencing of thousands to millions of DNA molecules simultaneously.
- This powerful tool is revolutionizing fields such as personalized medicine, genetic diseases, and clinical diagnostics by offering a high throughput option with the capability to sequence multiple individuals at the same time.
Thus Next Generation is a breakthrough procedure which added value in many medical sectors and changed the future of healthcare! To get more insights about Next Gen Sequencing, Join us on 14th-15th November 2019 in Munich, Germany for Future of Personalized Medicine Summit for Oncology by registering yourself from the link below :