Over last few years, demand for fast, cost effective and exact information of genome sequencing is rising for undeniably reinvigorating the fields like Human Genome Project and a broad range of biological phenomena (like genetic variation, protein DNA interaction, RNA expression, change in chromosomal conformation) and much more to go. Since Sanger sequencing (which is conventional sequencing method) has many disadvantages like High throughput results, High cost per base ($1000 per million bases), Long time to generate data, need for cloning, to overcome these challenges leads to the development of Next Generation Sequencing (NGS) technologies. The primary advantage provided by NGS is high throughput results in less time and in cost effective manner. Some of the applications are:
1. Whole- Genome Sequencing
Genome–wide association studies (GWAS) have been the most common approach for identifying disease associations across the whole genome. Whole genome sequencing means genome sequencing of the whole genome of an organism. NGS will detect rare variants and missing data which are not identified by microarray based studies.
2. Location of DNA –Binding Proteins
Identification of DNA-binding protein location is identified by ChIP and identification of binding loci of DNA binding protein is identified in by vivo NGS techniques.
3. Study of chromatin structure
MNase (an enzyme) digestion followed by sequencing with NGS technique to map genome-wide locations of nucleosomes.
4. Transcriptome sequencing
Transcriptome ( the sum total of all messenger RNA in a cell) sequencing has been used for applications like gene expression profiling.
5. Small RNA Profiling
Small RNA (length less than 200 nucleotides) sequencing studies with NGS technology contributed to the discovery of a small RNAs such as Piwi-interacting RNAs.
6. Molecular barcoding
Multiplex sequencing (more than one sample can be read in one run) of samples from multiple individuals.
7. NGS in clinical setting
Usefulness of NGS in clinical setting varies for different disorders and genetic counseling. Identification of the molecular etiology allows the clinician to guide subsequent pregnancies
Generally, NGS sequencing involves 3 major steps: DNA sample preparation, immobilization, and sequencing. DNA is fragmented using restriction enzymes (i.e. molecular scissors) or sonication followed by adapter ligation.
This DNA preparation is known as ‘Sequencing Library’. Next step is the addition of adapters which require anchoring sequencing library to a solid surface. This the common step in all sequencing techniques.
There are 4 major sequencing techniques of NGS:
- ILLUMINA SEQUENCING: Libraries are constructed and amplified. In this approach, forward and reverse primers are used which are linked to adapter sequences. This amplification takes on the flow cell. Now clusters are generated known as Cluster Generation. Clusters are now ready to sequence using bioinformatics software.
- 454 PYROSEQUENCING: Adapter carrying DNA is amplified using PCR (polymerase chain reaction). Generation of millions of clonally amplified sequencing templates on each bead. Load beads into picotiter plate with enzymes i.e. sulfurylase and luciferase and chemical APS. DNA polymerase adds one of the nucleotides at a time i.e. for eg adenine will be added at one time and no other nucleotide is added with adenine. The addition of nucleotide will generate pyrophosphate (ppi). Pyrophosphate combines with sulfurylase in the presence of APS will form ATP. ATP converts luciferin to oxy luciferase and light in the presence of luciferase enzyme. Light is detected by detectors.
- ION TORRENT: Proton / PGM sequencing: DNA is fragmented and ligated with adaptors and amplified by PCR on the surface of 3-micron diameter beads, known as Ion Sphere particles. Template beads are loaded on proton-sensing plates. Each of the 4 nucleotides is added sequentially. If such bases are incorporated, protons are released; then a signal is detected; this signal is directly proportional to the number of bases incorporated.
- SOLiD SEQUENCING: DNA sample involves fragmentation and ligation with ‘P1’ and ‘P2’ adapters. PCR is used for amplification of DNA fragments. SOLiD technology applies partially degenerate, fluorescently labeled, DNA octamers with dinucleotide complement sequence recognition core. These detection oligonucleotides are hybridized to the template and perfectly annealing sequences are ligated to the primer. After imaging, unextended strands are capped and fluorophores are cleaved.
NGS technologies have promising results by reducing the cost of DNA sequencing, transcriptome, and interactomes by many magnitudes so that researchers can extend their project on the large-scale due which NGS become a widespread technology.
1: Department of Biotechnology, Jamia Millia Islamia, Delhi, India
2: Division of Sustainable Technology, Rudraksh Prodhyogiki Sangathan, Delhi, India
3: Department of Biochemistry, Institute of Home Economics, University of Delhi