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Next Generation Sequencing Revolution: What makes Massive Parallel Sequencing so powerful?
In the field of biomedical sciences, the rapid development of DNA sequencing technology is constantly changing our understanding of the genome. Massive Parallel Sequencing, also known as Next-Generation Sequencing (NGS), is one of the core technologies of this change.
Massive Parallel Sequencing enables us to generate from 1 million to 43 billion short sequence reads in a single device run, something that was unimaginable in the past.
From 1993 to 1998, several high-throughput DNA sequencing technologies were developed and commercialized after 2005. These techniques take advantage of miniaturized and parallelized platforms, allowing large amounts of DNA sequence data to be obtained per run. Compared with traditional Sanger sequencing, Massive Parallel Sequencing can complete sequencing on a larger scale and no longer relies on independent sequencing reactions.
Operation process of NGS platform
For commercially available NGS platforms, the basic process of DNA sequencing usually includes the following steps. First, DNA sequencing libraries are generated by in vitro PCR clonal amplification; second, sequence determination is performed by synthesis; and finally, these spatially separated amplified DNA templates are sequenced in parallel without the need for physical separation steps. This parallelized reaction model enables the generation of hundreds of trillions to billions of nucleotide sequences per run, which greatly enriches the available sequence data.
Massive Parallel Sequencing not only provides higher data output, but also significantly reduces sequencing costs, which are now close to US$1,000 per genome.
Template preparation method
Two methods are used to prepare templates in NGS reactions, one is amplification template from a single DNA molecule, and the other is using a single DNA molecule template directly.
Cream PCR method
In the cream PCR method, a DNA library is first generated, and then single-stranded DNA fragments are attached to the surface of beads, which are encapsulated within water-oil cream particles to form a PCR microreactor to amplify a single DNA template.
Bridge amplification method
Bridge amplification is achieved by covalently attaching forward and reverse primers to the surface of the flow cell. This technology is widely used to create high-density template clusters for NGS, suitable for subsequent sequencing reactions.
Sequencing method
There are several main NGS sequencing methods, including Sequencing by Synthesis, Pyrosequencing, and Sequencing by Reversible Terminator Chemistry.
The principle of synthetic sequencing relies on the endonucleoside digestion of DNA polymerase and determines the sequence of the sample by detecting the incorporation of each nucleotide. This technology has been adopted by almost all parallel sequencing instruments.
Flame sequencing combines solid support materials and engineered DNA polymerases to detect each nucleotide addition through instant luminescence. The development of these technologies has made DNA sequencing faster, more accurate and more efficient.
Future trends and challenges
As technology continues to advance, the cost of NGS continues to decrease and its application range becomes wider and wider. However, the technology still faces some challenges, including high dependence on instruments and the complexity of data analysis. Future research may focus on how to further improve the reading accuracy and speed, and how to simplify the data analysis process.
How will Massive Parallel Sequencing further advance our understanding of life sciences in future genome research?
In this genomics revolution, how will the potential of Massive Parallel Sequencing affect our future?
