DNA sequencing, allows the sequencing of nucleotide and hence genes within a biological genome, and has been initially commercialized as the Sanger Technology. Next-generation sequencing (NGS), massively parallel or deep sequencing are related terms that describe a DNA sequencing technology that has revolutionized genomic research. Using NGS an entire human genome can be sequenced within a single day. In contrast, the previous Sanger sequencing technology used to decipher the human genome, required over a decade to deliver the final draft. Although in genome research NGS has mostly superseded conventional Sanger sequencing, it has not yet been translated into routine clinical practice.
With advancing knowledge of clinical symptoms and diseases, technology is increasing its speed for man to be able to diagnose faster and more accurately. Hence, the need for cost efficiency also crops up. With NGS, the decreasing cost and increasing capacity of DNA sequencing has led to vastly increased opportunities for population-level genomic studies to discover novel genomic alterations associated with both Mendelian and complex phenotypes. Precision medicine offers patients with critical complex diseases like cancer, a chance for survival, through tailor-made medications and not the usual generic ones. NGS has a lot to offer and there are many modifications of the instrumentation and technique used.
Pyrosequencing
In pyrosequencing, the sequencing reaction is monitored through the release of the pyrophosphate during nucleotide incorporation. A single nucleotide is added to the sequencing chip which will lead to its incorporation in a template-dependent manner. This incorporation will result in the release of pyrophosphate which is used in a series of chemical reactions resulting in the generation of light. Light emission is detected by a camera which records the appropriate sequence of the cluster.
Sequencing by Synthesis
Sequencing by synthesis utilizes the step-by-step incorporation of reversibly fluorescent and terminated nucleotides for DNA sequencing. The nucleotides used in this method have been modified in two ways: 1) each nucleotide is reversibly attached to a single fluorescent molecule with unique emission wavelengths, and 2) each nucleotide is also reversibly terminated ensuring that only a single nucleotide will be incorporated per cycle. The fluorescent signal is read at each cluster and recorded; both the fluorescent molecule and the terminator group are then cleaved and washed away.
Sequencing by Ligation
Sequencing by ligation relies on short oligonucleotide probes that are ligated to one another. These oligonucleotides consist of 8 bases (from 3’-5’): two probe specific bases (there are a total of 16 8-mer probes which all differ at these two base positions) and six degenerate bases; one of four fluorescent dyes are attached at the 5’ end of the probe. The sequencing reaction commences by binding of the primer to the adapter sequence and then hybridization of the appropriate probe. This hybridization of the probe is guided by the two probe specific bases and upon annealing, is ligated to the primer sequence through a DNA ligase.
Ion Semiconductor Sequencing
Ion semiconductor sequencing utilizes the release of hydrogen ions during the sequencing reaction to detect the sequence of a cluster. Each cluster is located directly above a semiconductor transistor which is capable of detecting changes in the pH of the solution. During nucleotide incorporation, a single H+ is released into the solution and it is detected by the semiconductor.
Different NGS Platforms:
1. Illumina (Solexa) sequencing
Illumina sequencing works by simultaneously identifying DNA bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain.
2. Roche 454 sequencing
This method is based on pyrosequencing, a technique that detects pyrophosphate release, again using fluorescence, after nucleotides are incorporated by polymerase to a new strand of DNA.
3. Ion Torrent: Proton / PGM sequencing
Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase and therefore differs from the previous two methods as it does not measure light.
Applications:
Next-Generation sequencing is a very useful and cost-effective technique. A few applications are as follows:
- Molecular Marker Discovery
- Transcriptome sequencing
- Allele mining
- Exome Sequencing
- Metagenomics
- Single cell genomics
- Protein Binding domain prediction
- Phylogenetic and ecological study
- Epigenetics
- Multiple Genome Sequencing
- Drug target identification and pharmaceutics
- Vaccinology
The discovery of Next Generation Sequencing has allowed one to solve the problem of large data set handling, turnover time and the surrounding cost. It has been extended in pharmacology and is used for determining personalized therapies for patients. There is a lot of research that is yet possible, for example fine tuning the reads, improving on the quality score, integrating interactomics to distinguish between individuals and cells and many more. However, NGS remains one of the specialized discoveries that has made culmination of genetics, ecology and therapy, easier.