NGS is a low-priced, speedy and high-throughput alternative to Sanger-method based DNA sequencing. It offers parallel-sequencing of millions of DNA fragments concurrently and allows an entire genome to be sequenced in a small time.
NGS is basically a three-step process, namely:
1). Template preparation
2). Sequencing and imaging
3). Data analysis.
All the platforms for NGS like, Roche 454, Illumina-MiSeq, Illumine HighSeq, ion-torrent, Nanopore-Sequencing follows the above mentioned steps but SMRT Single-Molecule real-time technology is an exception. SMRT also called as third-generation sequencing is a sequence-by-synthesis approach based on real-time imaging of fluorescently tagged nucleotides as they are incorporated into nascent DNA molecules from individual DNA templates. SMRT has several advantages over other NGS platforms when apply to small genomes like de novo assembly of novel genomes where amplification biases can lead to fragmented assemblies whenever a complex repeat or poorly amplified region is encountered which results in poor sequencing.
Since the datasets generated though NGS are very large, reading entire data is highly inefficient. To overcome this, most programs use these datasets as databases to easily retrieve specific sequence or piece of data. For the visualisation of genomic data,bioinformatics plays a crucial rule, many tools have been designed that can handle massive amount of data produced by NGS. Also, with the rapidly growing era of genomic studies, many software and algorithms are delibrately being developed to meet the need of high accuracy, speed, efficiency and ability to deal with large datasets.
Various applications of NGS involves Transcriptome characterization, Gene identification and expression analysis, Digital Transcriptomics, Candidate-gene finding, Whole genome sequencing, targeted sequencing, Large-scale identification and development of molecular markers, nucleotide-variation profiling, and epigenetics.
Two vastly used practical applications of NGS are:
Whole exome sequencing (WES): The aim of WES is to find genetic variants that alter the proteins sequence and can be used in the clinical diagnostics to analyze the cause of disease in a patient. Mutations in the gene coding and regulatory regions can give rise to rare genetic diseases with many possible causes. Sequencing only exome instead of whole genome will be cost-effective and consume less time to allow the clinicians and researchers diagnose the possible causes of the disease.
Target Sequencing: A powerful approach to detect variants and locate SNPs to target all types of variation across relevant genomic regions, including low complexity regions like repeat expansions, promoters, and flanking regions of transposable elements.
References:
1). Roberts RJ, Carneiro MO, Schatz MC. The advantages of SMRT sequencing. Genome Biology 2013 Jul 3;14(7):405
2). Bahassi EM and Stambrook PJ. Next-generation sequencing technologies: breaking the sound barrier of human genetics. Mutagenesis 2014 August 22;29(5):303
3). Grada A and Weinbrecht K. Next-Generation Sequencing: Methodology and Application. Journal of Investigative Dermatology 2013 August ;133(11):248