Next Generation Sequencing Techniques

Introduction  

The use of next-generation sequencing technologies to sequence the entire genome of an individual has led to revolutionary leaps in the understanding of human disease and evolution. It is these technologies that make it possible for us to understand the genome of every organism on the planet in unprecedented detail. From the early days when the human genome was first sequenced to the present day, there have been rapid improvements in the sequencing platforms and the data produced.

Next generation sequencing technologies can deliver large amounts of data at low cost and high precision, providing new opportunities for cancer research. Genome sequencing is now one of the most widely utilised tools for determining underlying genomic activity. Since 2005, newer genome sequencing technologies have opened up a new frontier in genomic study. This article summarises “next-generation” genomic sequencing approaches. Advanced sequencing technologies such as  454 sequencing, SOLiD DNA sequencing technology, DNA nanoball sequencing, massively parallel signature sequencing, polony sequencing, ion torrent technology are highlighted.

 Next generation sequencing technologies are now becoming more and more prevalent in the areas of DNA sequencing, RNA sequencing, and epigenetics. They allow researchers to sequence all of the DNA or RNA in a cell or tissue in a single experiment, rather than needing several experiments for each. These next-generation sequencing technologies employ high-throughput and lower costs, as well as higher accuracy.

The Nanopore Sequencing Technology

Nanopores are structures that are made from porous lipid bilayers. Nanopores are typically 20-200 nm in diameter. If a molecule passes through a nanopore, it causes changes in the electrical current that is measured. Nanopore sequencing is the latest and most promising technique for DNA sequencing. The technique is based on the fact that the structure of DNA is such that electric current can be used to determine the order of its nucleotides, or subunits. In nanopore sequencing, a protein that acts as a nanopore is inserted into a lipid bilayer. 

Using a nanopore sequencer, you are able to sequence DNA using nanopores which are extremely small electrical holes.These little holes are too small to be able to move an electron through. Because of this, you can sequence DNA by measuring the flow of positive ions (positive charge) through the hole. The holes are in a solid ‘chip’, which looks like a silicon wafer. Nanopore sequencing involves the sequencing of DNA by passing single-stranded DNA through a nanopore membrane, a tiny hole with a diameter of 10-15 nanometres. 

SOLID Sequencing technology

Sequencing by oligonucleotide ligation and detection (SOLID) is based on the ligation of synthetic DNA oligonucleotide probes to a target DNA sequence. Once a probe has hybridised to its target sequence, a nick is introduced into the target. Repair of the nick then allows the ligation of a short oligonucleotide, which contains a fluorescent reporter group, to the nick. Detection of the resultant fluorescently labelled target sequence by capillary electrophoresis (CE) allows quantitation of the target DNA.

Solid sequencing technology is utilised for sequencing DNA by fragmenting an unknown sequence into smaller pieces and then sequencing each piece separately. Solid sequencing could be used to discover the sequence of new species of unknown DNA or to sequence an unknown DNA from an environment. Most sequencing methods require amplification of the DNA to make it easy to read. Solid sequencing is a method of sequencing without amplification of DNA in order to determine the DNA sequence without knowing what the previous sequence is. 

Solid sequencing is an advanced new sequencing technology that has the potential to be the next generation of sequencing technology. Solid sequencing technology is based on the sequencing by ligation technology (SOLiD) developed by Applied Biosystems (ABI). SOLiD was the first commercial solid sequencing technique. It was released in 2002 and was initially used only for research purposes. 

The goal of the entire project was to sequence the whole genome of an uncharacterized organism. This was done using SOLID. SOLID is based on the use of artificial DNA probes, which are constructed to recognize a sequence of interest. Once a probe sequences its target, it is ligated onto a small oligonucleotide, which contains a fluorescent reporter group.

Conclusion 

Next generation sequencing technology is much more flexible because it uses single nucleotide resolution to detect where the sequence breaks and where the individual nucleotides are added, allowing for the insertion of an arbitrary length nucleotide into the sequence. Next-generation sequencing technology (NGS) is a type of DNA sequencing that uses the fact that DNA can be sequenced by placing a nucleotide at a time through a series of steps, ending with the fluorescently tagged nucleotide added to a nucleotide sequence on a solid support.