What is Next-generation sequencing (NGS)?

What is Next-generation sequencing (NGS)?

Next-generation sequencing (NGS) is defined as technology allowing one to determine in a single experiment the sequence of a DNA molecule(s) with total size significantly larger than 1million base pairs (1millionbp or 1Mb).

What is Next-generation sequencing (NGS)?

Next-Generation Sequencing (NGS) is a term used for describing a range of various modern sequence technology, also known as high-throughput sequencing. These technologies enable DNA and RNA to be sequenced much faster and cheaper than the Sanger sequence used before. As a result, the research in genomics and molecular biology was revolutionized Such technologies include the following:

What is Next-generation sequencing (NGS)?

Illumina (Solexa) sequencing

Illumina sequencing works by simultaneously identifying DNA bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain.

Roche 454 sequencing

This method is based on pyrosequencing, a technique which detects pyrophosphate release, again using fluorescence, after nucleotides are incorporated by polymerase to a new strand of DNA.

Ion Torrent: Proton / PGM sequencing

Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase and therefore differs from the previous two methods as it does not measure light.

What can Next-generation sequencing (NGS) do?

NGS technology has fundamentally changed the questions that can be asked and answered by scientists. A variety of applications are enabled by innovative sample preparation and analysis options. NGS allows scientists, for example:

  • Rapidly sequence whole genomes
  • Zoom in to deeply sequence target regions
  • Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants and splice sites, or quantify mRNAs for gene expression analysis
  • Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein interactions
  • Sequence cancer samples to study rare somatic variants, tumour subclones, and more
  • Study microbial diversity in humans or in the environment.

Key Highlights of NGS

  • There are two major paradigms in next-generation sequencing (NGS) technology: short-read sequencing and long-read sequencing. Short-read sequencing approaches provide lower-cost, higher-accuracy data that are useful for population-level research and clinical variant discovery. By contrast, long-read approaches provide read lengths that are well suited for de novo genome assembly applications and full-length isoform sequencing.
  • NGS technologies have been evolving over the past 10 years, leading to substantial improvements in quality and yield; however, certain approaches have proven to be more effective and adaptable than others.
  • Recent improvements in chemistry, costs, throughput and accessibility are driving the emergence of new, varied technologies to address applications that were not previously possible. These include integrated long-read and short-read sequencing studies, routine clinical DNA sequencing, real-time pathogen DNA monitoring and massive population-level projects.
  • Although massive strides are being made in this technology, several notable limitations remain. The time required to sequence and analyse data limits the use of NGS in clinical applications in which time is an important factor; the costs and error rates of long-read sequencing make it prohibitive for routine use, and ethical considerations can limit the public and private use of genetic data.
  • We can expect increasing democratization and options for NGS in the future. Many new instruments with varied chemistries and applications are being released or being developed.
What is Next-generation sequencing (NGS)?
Credit: abmgood An illustration of the similarities and difference between the different Next Generation Sequencing platforms.

Method of Next generation sequencing

  • Library preparation: libraries are created using random fragmentation of DNA, followed by ligation with custom linkers
  • Amplification: the library is amplified using clonal amplification methods and PCR
  • Sequencing: DNA is sequenced using one of several different approaches.

Library preparation

First of all, DNA is fragmented by sonication (excitement with ultrasound) or enzymatic to create smaller strands. Then, with the aid of DNA ligase, which join DNA strands, adjusters (short, double-stranded parts of synthesized DNA) are ligated to those fragments. The adapters allow the sequence to be attached to an additional component.

Adapters are synthesized to ‘stick,’ while one end is ‘blunt,’ so the other ends are ‘blunt,’ which is not cohesive. This could lead to the potential problem of base pairing and thus dimer formation between molecules. The chemical structure of DNA is used to prevent this since it is linked between the 3′-OH and 5′-P ends. The DNA ligas can not create a bridge between the two ends by removing the phosphate from the bold end of the adaptor and thus creating a 5′-OH end.

The library fragments must be classified spatially in PCR colonies or as conventionally known “polonies,” consisting of a lot of copies of a certain library fragment, in order to be successful. As these polonies are plated planarly, enzymatically parallel the features of the array can be manipulated. This library-building method is much quicker than the colony picking and E-working intensive procedure. Coli cloning for the isolation and amplification of DNA for Sanger sequence, however, is at the expense of the fragment read length.

Amplification

Library amplification is necessary so that the received sequencer signal is sufficiently strong to be accurately detected. In the context of enzyme amplification, certain library fragments may be preferentially amplified by the use of phenomena like ‘biasing’ and ‘duplication.’ Rather it is possible to use PCR to generate a great number of DNA clusters in different types of amplification process.

  • Emulsion PCR
  • Bridge PCR

Sequencing

Several competing methods of Next Generation Sequencing have been developed by different companies.

  • Pyrosequencing
  • Ion Torrent semiconductor sequencing
  • Sequencing by ligation (SOLiD)
  • Reversible terminator sequencing (Illumina)
  • 3′-O-blocked reversible terminators
  • 3′-unblocked reversible terminators

Application of Next generation sequencing

  • The next-generation sequence allows researchers to collect large amounts of genomic sequence data. This technique has a host of applications, such as diagnosis and understanding of complex diseases; genome sequence; analysis of epigenetic modifiers. mitochondrial sequencing; transcriptome sequencing – understanding how the altered expression of genetic variants affect an organism. DNA techniques have been used to identify and isolate genes responsible for certain diseases and provide the correct copy of the defective gene known as ‘gene therapy’.
  • One major focus of gene therapy is cancer – a potential way would be to introduce an antisense RNA into the oncogene, which is triggered to form tumour cells, which specifically prevent the synthesis of a targeted protein. Another method is called suicide gene therapy that introduces genes to selectively kill cancer cells. A large number of genetic codes are known for toxic proteins and enzymes, which would cause death in cells. The problem is ensuring a highly accurate supply system to prevent the killing of healthy cells.
  • These approaches are enabled by sequencing tumour genomes, enabling medical experts to more effectively tailor chemotherapy and other cancer treatments to the unique genetic composition of their patients and to revolutionize the diagnostic stages of personalized medicine.
  • The cost of DNA sequencing will decrease, and this will lead to a number of problems. Sequencing generates enormous amounts of data and the processing and storage of data involvement pose many computational challenges. Ethical issues are also present, like ownership of a DNA in sequencing the DNA. DNA sequencing data must be stored securely because insurance groups, mortgage brokers and employers are concerned that the data can be used to alter insurance quotes or to differentiate between candidates.
  • Sequencing may contribute to finding out if a person has an increased risk of developing a certain condition. However, it is another problem whether the patient is being advised or if the disease is cured.

What is Next-generation sequencing (NGS)?

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