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- Real-time PCR
- What is Next Generation DNA Sequencing?
- RNA sequencing
- Biological interpretation of gene expression data
- Genotyping, epigenetic and DNA/RNA-protein interaction methods
- DNA/RNA-protein interactions
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What is Next Generation DNA Sequencing?
In contrast to microarray methods, sequence-based approaches directly determine the nucleic acid sequence of a given DNA or cDNA molecule.
The first major foray into DNA sequencing was the Human Genome Project. This project, which used first-generation sequencing, known as Sanger sequencing (the chain-termination method), took 13 years, cost $3 billion and was completed in 2003.
Compared to conventional Sanger sequencing using capillary electrophoresis, the short read, massively parallel sequencing technique is a fundamentally different approach that revolutionised sequencing capabilities and launched the second-generation sequencing methods – or next-generation sequencing (NGS) – that provide orders of magnitude more data at much lower recurring cost.
Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such revolutionised the study of genomics and molecular biology. Such technologies include:
Advantages of NGS
NGS can be used to analyse DNA and RNA samples and is a popular tool in functional genomics. In contrast to microarray methods, NGS-based approaches have several advantages including:
- a priori knowledge of the genome or genomic features is not required
- it offers single-nucleotide resolution, making it possible to detect related genes (or features), alternatively spliced transcripts, allelic gene variants and single nucleotide polymorphisms
- higher dynamic range of signal
- requires less DNA/RNA as input (nanograms of materials are sufficient)
- higher reproducibility
- 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
- Roche 454 sequencing: This method is based on pyrosequencing, a technique which detects pyrophosphate release, again using fluorescence, after nucleotides are incorporated by polymerase to a new strand of DNA.
- 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.
The next few pages provide step-by-step explanations of how each of these NGS technologies work.
These Youtube videos give a useful overview of how Sanger sequencing and the different next generation sequencing technologies work and when to use them: