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The Past of Sanger Sequencing and the Future of NGS: How does DNA sequencing go from single to parallel?
In the field of biomedical science, DNA sequencing technology has undergone significant changes.Since the introduction of Sanger sequencing, the mystery of the genome has been unveiled for us, and the emergence of next-generation sequencing (NGS) has pushed this technology to new heights.This technological leap from single to parallel not only increases the speed of sequencing, but also reduces the cost of sequencing, thus changing the research landscape of genomics.
The emergence of NGS allows us to obtain tens of millions of genomic data in a short period of time, which is incomparable to Sanger sequencing.
NGS refers to various high-throughput DNA sequencing methods that utilize the concept of large-scale parallel processing.Many of these technologies emerged one after another from 1993 to 1998 and were commercialized in 2005.These technologies allow each instrument to generate from 1 million to 43 billion short sequence reads when run.In these platforms, technical configurations and sequencing chemistry differ, but they share a common technical paradigm: large-scale parallel sequencing through spatially isolated, clonally amplified DNA templates or single DNA molecules.
At the same time, Sanger sequencing is also known as first-generation sequencing, which relies on electrophoresis to separate the end products.Although this method has a long history, it seems unscrupulous when facing the current scientific needs.NGS methodology allows us to complete large-scale sequencing work at one time, bringing unprecedented efficiency and accuracy to genetic research.
NGS platform
The process of DNA sequencing using commercially available NGS platforms is usually divided into several steps:
- DNA sequencing libraries were generated by in vitro PCR clonal amplification.
- DNA sequence is determined by synthetic sequencing based on the increase of nucleotides on the complementary strand.
- Spatially isolated, amplified DNA templates are sequenced simultaneously in a large-scale parallel manner, without the need for physical separation steps.
Although these steps are similar on most NGS platforms, the strategies of each platform vary, which makes each technology unique and application areas in the market.
The parallelized sequencing reaction of NGS can generate hundreds of mega to gigabit nucleotide sequence reads in a single instrument run.This change greatly increased the amount of available sequence data, resulting in fundamental changes in the genome sequencing methods of medical science.With the emergence of emerging NGS technologies and instruments, the cost of sequencing has also been significantly reduced, approaching the standard of only $1,000 per genome.
Template preparation method
When performing NGS reactions, there are two main methods for preparing templates: amplification templates from a single DNA molecule and a single DNA molecule template.
For imaging systems that cannot detect a single fluorescent event, the DNA template needs to be amplified.Common amplification methods include emulsion PCR, rolling ring amplification and solid phase amplification.
Lotion PCR
In the emulsion PCR method, a DNA library is first generated by random fragmentation of genomic DNA, and then the single-stranded DNA fragment is connected to the surface of the particle with the linker. After the sock is opened, each particle represents a DNA fragment.The particles are then packaged into water-oil emulsion droplets, each of which is a PCR microreactor, where an amplified single DNA template copy is produced.
Bridge Amplification
In bridge amplification, the forward and reverse primers are covalently attached to the substrate of the flow cell at high density.After the enzymatically extended reagent is exposed, the paired template extends over the surface primers, ultimately completing the local amplification of DNA polymers at millions of different locations, becoming an independent template cluster.
Single-molecular template
The preparation of single-molecule templates is more intuitive in comparison, which does not require a PCR step.Generally, single-molecular templates are fixed on solid support and amplified in different ways.As in the first approach, the spatially distributed primers are immobilized on the solid support, and the randomly cleaved DNA fragments are then hybridized to the immobilized primers.
Sequencing method
The sequencing methods of NGS have their own characteristics, including synthetic sequencing, pyrosequencing and reversible terminator chemistry.The purpose of synthetic sequencing is to determine the sequence of the sample by detecting the addition of nucleotides of DNA polymerase.
Arguably, after losing the tedious steps required for traditional Sanger sequencing, NGS provides a more efficient paradigm for genomic research.
As the technology develops, we have witnessed the transition of DNA sequencing from an early single-sequence method to today's parallel processing model, which not only greatly improves efficiency, but also brings unprecedented advantages in cost and time.In which direction will the future DNA sequencing technology develop?
