DNA sequencing is the process of determining the sequence of nucleotides within a DNA molecule. The first of these sequencing technologies is known as Sanger sequencing. The advent of new techniques and methods has led to rapid progression and given rise to different generations of sequencing technologies.
First Generation Sequencing -
Sanger sequencing Sanger Sequencing is known as the chain termination method or the sequencing by synthesis method. It involves chemically modified nucleotides called dideoxy-nucleotides (dNTPs) that incorporate into the DNA strand and allow fragments to be separated based on their size.
Maxam-Gilbert method is also known as the chemical degradation method. It relies on the cleaving of nucleotides by chemicals and is most effective with small nucleotides polymers. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of the four reactions (C, T+C, G, A+G). This reaction leads to a series of marked fragments that can be separated according to their size by electrophoresis (1).
Second generation sequencing (SGS) -
The basic characteristics of second generation sequencing technology are: (1) The generation of many millions of short reads in parallel, (2) The speed up of the sequencing process compared to the first generation, (3) The low cost of sequencing and (4) The sequencing output is directly detected without the need for electrophoresis.
Roche/454 sequencing is pyrosequencing technique, based on the detection of pyrophosphate released after each nucleotide incorporation in the new synthetic DNA strand. The pyrosequencing technique is a sequencing-by-synthesis approach. The Roche/454 method is able to generate relatively long reads which are easier to map to a reference genome. The main errors detected of sequencing are insertions and deletions due to the presence of homopolymer regions. (1), (2)
Ion torrent sequencing
Life Technologies commercialized the Ion Torrent semiconductor sequencing technology in 2010. It is similar to 454 pyrosequencing technology but it does not use fluorescent labeled nucleotides like other SGS technologies. It is based on the detection of the hydrogen ion released during the sequencing process, which changes the pH of the solution. This change is detected by a sensor and converted into a voltage signal which is proportional to the number of nucleotides incorporated .The Ion Torrent sequencers are capable of producing read lengths of 200 bp, 400 bp and 600 bp with throughput that can reach 10 Gb for ion proton sequencer. The major advantages of this sequencing technology are focused on read lengths which are longer than other SGS sequencers and fast sequencing time between 2 and 8 hours. (3)
Other SGS technologies include Illumina/Solexa sequencing which is sequencing by synthesis approach and is currently the most used technology in the NGS market. Another method is ABI/SOLiD sequencing. Supported Oligonucleotide Ligation and Detection (SOLiD) is a NGS sequencer marketed by Life Technologies, and which has very high accuracy (4).
Third Generation of Sequencing (TGS)
TGS technologies offer a low sequencing cost and easy sample preparation without the need for PCR amplification in an execution time significantly faster than SGS technologies. In addition, TGS are able to produce long reads exceeding several kilobases for the resolution of the assembly problem and repetitive regions of complex genomes. The most widely used TGS technology is single molecule real time sequencing approach (SMRT). The sequencers that use this are Pacific Biosciences and the MinION sequencer from Oxford Nanopore Technology (ONT).
Next Generation Sequencing (NGS) Technologies
Next generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day. (5)
- Kchouk M, Gibrat JF, Elloumi M (2017) Generations of Sequencing Technologies: From First to Next Generation. Biol Med (Aligarh) 9395. doi:10.4172/0974-8369.1000395
- Behjati, Sam, and Patrick S. Tarpey. "What is next generation sequencing?." Archives of Disease in Childhood- Education and Practice 98.6 (2013): 236-238.