EVERY ORGANISM on the earth whether it is bacteria virus, plants, animals and even human possess its information in the form of DNA, which is referred as genetic information. DNA is deoxyribonucleic acid consists of four nucleotide bases Adenine (A), Guanine (G), Thymine (T) and Cytosine (C). A fifth pyrimidine nucleobase, Uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. Uracil is not usually found in DNA, occurring only as a breakdown product of cytosine. This chemistry of DNA is same in all species. DNA is responsible for phenotypic (physical appearances) and genotyphic (genetic information) characteristics of the organisms. After the discovery of the DNA structure, the major problem to face by the scientist through out the world was how can we read the sequence of the DNA? The scientists started to explore the methods of reading the DNA, what is now called the DNA sequencing.
The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography, following the development of dye-based sequencing methods with automated analysis. To have clear understanding of DNA sequencing lets imagine the string of beads with different colors like red, blue, black and white. The counting of these color beads with sequence wise from one end to another is similar example for the DNA sequencing, where the color beads are replaced with A (Adenine), T (Thiamine), G (Guanine) and C (Cytosine).
Chain termination method or dideoxy method
This was the first method proposed by Frederick Sanger in early 1970’s. The key principle of the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. The dideoxynucleotide during the elongation reaction of DNA synthesis terminates the reaction. The individual tube was visualized by gel with resolution of one nucleotide. As the gel separates the DNA fragments as single base, thus the nucleotide can be counted from bottom to top with decreasing the length of DNA fragments.
First Generation Sequencing (FGS)
With the advancement of time the Sanger method was modified with automatic detector system. Instead of dideoxynucleotides here they used dideoxynucleotides (labled with colored fluorescent dye). In this method instead of using four individual tubes, the DNA sample is analysed in single tube and chain termination takes place with dideoxynucleotides which is detected by fluorescence detector laser. The automated Sanger sequencing method is considered as a first generation technology and newer methods are referred to as next generation sequencing.
Next Generation Sequencing (NGS)
The number of latest technologies has been developed and referred as NGS; the NGS is broad terminology which included many technologies itself.
The NGS described here is mainly categorized as follow Template preparation, Sequencing, and Imaging and data analysis
Template preparation: Usual trend in template preparation (DNA) for sequencing is the chopping of DNA in to small pieces. In most of the cases the template is fixed or immobilized on solid surface. Millions of templates DNA are allowed for rapid sequencing at the same time. A number of NGS technology used template preparation in different ways like clonally amplified and single molecule.
Sequencing and imaging: Template preparation mostly composed of clonally amplified and single molecule templates. The template from these methods are further processed for sequencing and imaging using the Cyclic Reversible Termination (CRT), Sequencing By Ligation (SBL), Single Nucleotide Addition (SNA) also called Pyrosequencing, and Real Time Sequencing (RTS).
In CRT method the DNA polymerase elongates the lagging strand of DNA and incorporates the terminator nucleotide and thus DNA synthesis terminates. Another cleavage step is applied where the terminating group is removed and DNA synthesis continues again to amplify the remaining fragment.
The SBL is similar to CRT; here they use DNA ligase for joining the terminating fragment with new primers or probes to continue the DNA synthesis.
The SNA or Pyrosequencing do not rely on electrophoresis but is based on bioluminescence generated by pyrophosphate liberated during extension step. Upon incorporation of the complementary dNTP, DNA synthesis is reinitiated following the addition of the next complementary dNTP in the dispensing cycle. The order and intensity of the light peaks are recorded as flowgrams, which determines the underlying DNA sequence.
Pacific Biosciences is current pioneer of RTS technology. Unlike reversible terminators, real time nucleotides do not halt the process of DNA synthesis. The principle of RTS is the direct incorporation of dye labeled dNTP, followed by imaging using zero-waveguide detectors.
Genome Alignment and Assembly (GAA)
After sequencing and NGS data has been generated, the step of GAA and other bioinformatics approaches is needed. The GAA method can be carried out either compared with known reference or can be assembled using De novo assembling strategies. The decision to use either strategy is based on the intended biological application as well as cost and time considerations.
For example for the identification of genetic variation in specific species such as bacteria or Arabidopsis plant. This GAA performed under reference sequence is cheaper for Spleen Necrosis Viruses (SNVs) identification. One of the clear limitations of the reference sequence method is lack of certain repetitive regions in reference genome, which results in formation of gaps during alignment.
Another approach for GAA is assembly of genomes by using De novo genome assembly. The method does not rely on reference genome but compiled the newly generated data with sophisticated manner. The De novo assemblies have been reported for bacterial genomes and mammalian bacterial artificial chromosomes.
The writer is a research fellow at Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.