5.1 Next Generation Sequencing:
It is a method to determine the order of nucleotide in DNA and, as the name suggests, it applies to genome (complete set of DNA present in an organism/individual) sequencing, transcriptome profiling, DNA-protein interaction and epigenome characterization. Since 1977 Sanger sequencing (based on chain termination method) had been the primary choice for such purposes, because of its high affinity and low radioactivity but the major disadvantage in sanger sequencing is that it is time consuming and costly. The NGS technologies are different from the Sanger method in aspects of massively parallel analysis, high throughput, and reduced cost. Sequencing technologies include a number of methods that are grouped broadly as template preparation, sequencing approaches and imaging.
5.1.1 ) Template preparation methods for NGS:
Template for NGS is the DNA molecule whose sequence is to be determine. It can be used directly or can be processed depending on the type of imaging system it is applied to, as most imaging system have been developed to read multiple fluorescent events so amplified DNA fragment is used as a template for such systems. Thus two types of samples can be used as template for NGS reactions:
A. Single DNA molecule.
B. Amplified templates originating from single DNA molecules.
A common theme among NGS technologies is that the template is attached or immobilized to a solid surface or support. The two most common amplification methods are emulsion PCR (emPCR) and solid-phase amplification.