Genome sequencing is one of the most important fields in science and many research were done in this field especially in human genome. Genome is biological heredity information in building and maintaining living organisms. Genome is encoded in deoxyribonucleic acid (DNA) which in discrete units known as gene (Brooker, 2011). There are various types of DNA which is nuclear DNA, mitochondrial DNA (mtDNA) and chloroplast DNA. This essay will be focusing on DNA sequencing of nuclear DNA for human. Genome sequencing involve process known as DNA sequencing which process of reading nucleotide bases - adenine (A), guanine (G), cytosine (C) and thymine (T) in DNA (Bowman and Sanders, 2012). As genomics DNA uniquely described a species and an individual, DNA sequencing become an important method for scientist to do research on various applications such as evolutionary studies, finding and targeting genes, cloning and many more.
2.0 First-generation Sequencing
DNA sequencing technologies was first developed by Frederick Sanger, Walter Gilbert and Allan Maxam in 1970s which known as first-generation sequencing (Bowman and Sanders, 2012). First-generation sequencing involves two methods which are the chain-termination method developed by Sanger and the chemical sequencing method developed by Gilbert and Maxam. These technologies consist of three basic stages; preparations of sample, physical sequencing and genetic assemble (Schadt et al., 2010). Sample preparation involved cloning using polymerase chain reaction (PCR) to get template fragment, the template than purified and cut into fragments with different sizes and generated form same starting point. The process continue with physical sequencing stage using chain-termination method where the fragments are terminated using energy transfer and labeled with one of the four fluorescent dye terminator and DNA polymerase. All of the fragments will undergo capillary electrophoresis and fluorescence detection which produce the DNA sequence. Each of the four dyes corresponds accordingly to four different bases. However, this process produces low throughput with high cost and time.
3.0 Second-generation Sequencing
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Second-generation sequencing also known as next-generation sequencing (NGS) and these technologies are developed in 2000s. The first commercially technology was Roche 454 released in 2005. Second-generation sequencing also consist of three stages which are preparation of sample, sequencing and imaging, followed by genetic assemble and alignment (Metzker, 2010). Most of second-generation sequencing methods involve determination of DNA sequence by synthesizing complementary DNA from DNA template known as sequencing by synthesis (SBS). Preparation of sample or template usually did using two methods; single DNA templates and clonally amplified template from single DNA.
3.1 Roche 454 FLX System
Roche 454 FLX system was released in 2005 by 454 and in 2007, 454 was bought by Roche (Liu et al., 2012). In 2008, they released new technology 454 GS FLX Titanium system. This system use pyrosequencing technology which detect releasing of pyrophosphate during nucleotide incorporation. Template preparation in Roche 454 FLX system is done using clonally amplified template method which produces around 100 to 200 millions template. In this method, the DNA template is in cell free process and the adaptor that contain universal primer site will be ligated to the target ends which denatured the template into single strands DNA. Then, the DNA will be amplified using common PCR primers and will be connected to beads under rule of one DNA per beads (Metzker, 2010). The template will undergo emulsion PCR and the templates are put in the PicoTiterPlate (PTP) well. Each of the bases in the templates strand will correspond to the one of the dNTP and produce different colour of fluorescent light. This step was assisted by addition of luciferase, sulferylase and luciferin (Liu et al., 2012). The light generated will be detected by CCD camera under the slide and produce the DNA sequence. This technology can read approximately 230-400 bases. The advantage of this method is high throughput among the second-generation sequencing and fast run time. The limitations are high reagent cost, expensive machine and high error in homopolymer repeats. However, the cost is still relevant and affordable.
3.2 Ilumina/Solexa GA/HiSeq 2000 system
This system is the most widely used in DNA sequencing field. In 2006, Ilumina launched the Genome Analyzer (GA) and in the following year, the company bought by Solexa. In 2010, Solexa released HiSeq 2000 system which has the same sequencing strategy as GA with a few improvements. Ilumina/Solexa GA/HiSeq 2000 system involves preparation of template using clonally amplified template method, specifically solid-phase amplification and also uses the SBS technology. In the template preparation stage, randomly distributed clusters are produced using mate-pair templates through bridge amplification. The templates are amplified using PCR and the templates are denatured into single strand. The clonally amplified clusters are put on the slide. Next, the samples will undergo four colour cyclic reversible termination (CRT) process. The slide is flooded with reagent, the nucleotide bases are incorporated into DNA strand with different florescent dye and reversible blocked group for each base and. The excess reagent is washed out and followed by imaging to determine incorporated bases. Then, the newly incorporated bases are cleaved and wash again. This cycle is repeated until no reaction available. Each bases label with different dye corresponds to the template and produce different signal and detected by CCD. HiSeq 2000 contains two lasers and four filters to detect the signal. This technology can read approximately 2-150 bases. The advantage of this technology is very high throughput and the limitation is expensive and low multiplexing ability.
4.0 Third-generation Sequencing
4.1 PacBio RS system
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This system was released by Pacific Boiscience around 2010. This system use single-molecule real-time (SMRT) technique. In this technology, DNA synthesis process is not halt instead the imaging of incorporation of dye-labeled nucleotide onto the complementary template continue during the DNA synthesis process. Thus, this can be measured directly from DNA polymerase. The florescent dye is attached not to the base but to the phosphate chain. This system contains many SMRT Cells which each have zero-mode waveguide detectors (ZMW) nanostructure. The DNA polymerase is confined to the ZMW and the florescent signal is detected using gamma-labeled phosponucleotides. The ZMW only allow the florescence detection at the bottom of the glass and the DNA polymerase will continued with the primer in order to be read at the bottom of ZMW. DNA polymerase will release the dye naturally when it cleaves the phosphate chain and flooded the ZMW array. The fluorescence dye emit coloured light correspond to specific base and this will be detected by the instrument. The advantages of this technology are cheaper and faster run time. This is because SMRT technique only need minimal reagents and sample preparation, Besides, PCR amplification step not needed in this technology which lead to rapid process and prevent amplification bias. The other advantage is longer read lengths which is approximately more than 1000 bases which is due to maximum used of DNA polymerse. The limitation of this technology is low pass accuracy as this technology is new and still under development. Although this technology rapid, large scale and cost-effective, the second-generation sequencing is still widely used compared to this technology.
4.2 Nanopore Sequencing
This technology was started developed in 1995 but until now, the development of this technology still continues. Nanopore is small holes that use to attract DNA and DNA will pass through the nanopore. The principle is when the nanopore is immersed into conduction reagent, current will be will be create due to different potential around the nanopore cause by movement of ions. The current produce varied depends on the size and shape of the nanopore. There is various ways than can be used in nanopore sequencing. The nanopore can be made using alpha hemolysin, Mycobacterium smegmatis porin A (MspA) and may contain florescence. This technology will give various advantages as the DNA can be directly sequenced using nanopore without the PCR amplification step, the labeling step using chemical and the optical detector method. In 2008, Oxford Nanopore Technologies licensed the nanopore sequencing technologies which use protein nanopore and used the synthetic bilayer to create ion movement which lead to production of current. The advantages of this technology are rapid, large scale and cost-effective as this technology exclude many process and can directly sequencing the DNA. The limitation is this technology is still under development and unknown accuracy.
DNA sequencing field undergo enormous technological transition due high demand for rapid, high throughput and low cost for DNA sequencing process. DNA sequencing will continue to developed especially the third-generation sequencing and this will lead to various new findings and invention for better future. Second-generation sequencing is still widely used due low accuracy of third-generation sequencing technology.