Next-generation sequencing: an overview for dummies

Next-generation sequencing: an overview for dummies

The Next Generation Sequencing (NGS) is on the verge of finally taking over after years of Sanger sequencing in molecular diagnostics as the gold standard. NGS is also referred to as high-throughput sequencing, since this allows many fragments to be sequenced in parallel (that is not possible by traditional Sanger sequencing).

There are several NGS systems developed by various companies. However, all of these systems have at least three fundamental steps in common: the development and the immobilisation of the DNA of the so-called sequence library, as well as the amplification and sequencing. In short, the next generation sequence is therefore carried out by:

1. preparation of the sequencing library

2. amplification

3. sequencing

Introduction to next generation sequencing

DNA PREPARATION & IMMOBILIZATION (i.e. creation of the sequence library)

By a random fragmentation process, the DNA sample is prepared. The obtained fragments are added to predefined sequences (called adapters) which are necessary to anchor and immobilise the DNA on support for sequencing. The so-called sequencing library consists of DNA fragments prepared by the addition of adapters.

The sequencing library is prepared by at least three various adapter types, i.e. by the linear adapter, circular adapter and bubble adapter. Of course, different kinds of anchor systems are also available. For example, fragments on a glass plate are attached to the SOLiD system.

AMPLIFICATION

Amplification can be done in emulsion or in solution (Roche and SOLiD systems). For instance, sequencing library fragments in the Roche FLX GS system together with so-called enrichment beads (small balls to which adapters can bind) are integrated into a microscopic bubble of water.

In these aqueous microbubbles, the amplification reaction (PCR) is carried out, where the DNA fragments are amplified multiple times. Clonal copies of the fragments attach to the entire enrichment bead’s surface. The enriching beads are then placed on the so-called Picotiter, supporting the sequencing reaction.

SEQUENCING

Sequencing reactions occur through extremely complex mechanisms based on microlitric fluidic systems, which control the flow of reagents that react with immobilised DNA. The reaction is based on fluidic systems.

In the single nucleotide interrogation systems* the immobilised DNA is mixed with a single nucleotide solution for each sequence cycle (which is integrated in addition to the sequence). The sequencing support is then washed off and the DNA mixed with a new nucleotoid-containing solution.

The event is registered by the machine when the nucleotide is added. It is also possible to register using molecular imaging systems (such as in the GS FLX system from Roche where the nuclear reaction occurs in combination with the emission of the flash of Light, which is the name of pyro-sequencing, from ancient Greek pyrographs, which means fire).

The registration of the incorporation event in the Ion Torrent method (Life Technologies) is focused on the detection of emissions of hydrogen ions emitted during the reaction of the polymerisation (semiconductor method).

As stated earlier, NGS systems are also known as high-throughput because fragments of DNA can be sequenced in parallel.

Note: Every nucleotide is tested individually in the GS FLX pyrosequencing system and in the Ion Torrent system (Life Technologies). All four nucleotides are studied in parallel in Illumina systems. In comparison, the four nucleotides in the Illumina systems are interrogated simultaneously.

Next-generation sequencing: an overview for dummies

Sakshi Sharmahttps://bioinformaticsindia.com
I am a Managing Partner at Bioinformatics India where I write blogs, look after all the partners, and manages the affiliates associated with the website.

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