Overcome technical difficulties and improve detection quality—Introduction to third-generation gene sequencing technology and related products

High-throughput sequencing technology (also known as next-generation sequencing technology) is used for single nucleotide polymorphism (single nucleotide polymorphism, SNP) or a small fragment sequence (usually less than 50bp in length) at a certain position in the genome.
Deletion (insertion-deletion, InDel) detection is more accurate, but it is very difficult to detect large structural variants
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The third-generation sequencing technology is marked by no PCR amplification and long read length, and realizes the individual sequencing of each DNA molecule, so it is also called single-molecule sequencing technology
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At present, the third-generation sequencing platform for long-read sequencing has been commercialized and widely used in scientific research
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Continuous upgrading of sequencing technology In 2008, Helicos BioSciences launched the world's first single-molecule sequencing platform, HeliScope, but the product has a short sequence read length and a high overall sequencing error rate
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Subsequently, long-read sequencing technologies for single molecules emerged
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At present, the long-read sequencing platform refers to the technology platform whose single-molecule sequencing length is not less than KB level
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Today, the commercialized long-read sequencing platforms mainly include the single molecule realtime sequencing (SMRT) platform of Pacific Biosciences and the nanopore sequencing (Nanopore) platform of Oxford Nanopore Technologies
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The third-generation sequencing technology developed by Pacific Biosciences, a single-molecule real-time sequencing platform, is called SMRT.
This technology is based on two important inventions and effectively overcomes the major problem of sequencing read length in the field of sequencing.

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Among them, the zero-mode waveguides (ZMWs) technology can limit the excitation light to a certain range at the bottom of the single-molecule nanopore, effectively filtering the background noise; On the group, it can help DNA polymerase to complete an all-natural DNA chain synthesis process
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Specific products based on this principle include PacBio Sequel sequencer and PacBio Sequel II sequencer
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The PacBio Sequel sequencer is the first third-generation sequencing technology product to achieve commercial application, which breaks through many technical bottlenecks of traditional short-read sequencing
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PacBio Sequel II sequencer is an upgraded version of PacBio Sequel, which can provide two sequencing modes, CLR Library and CCSlibrary (HIFI).
The PacBio Sequel sequencer is an 8x improvement
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At the same time, the single-base accuracy of CCS reads (sequencing fragments) has also been greatly improved.
After 4 times of sequencing, the accuracy of the same fragment can reach about 99%
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Nanopore single-molecule sequencing platform Nanopore is a technology based on electrical signal sequencing.
The principle is to drive single-stranded nucleic acid molecules through nano-sized protein pores through electric field force.
Therefore, the base information on each nucleic acid molecule can be identified according to the current signal, thereby realizing the sequencing of single-stranded nucleic acid molecules
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Because the principle of this sequencing technology is quite different from other platforms, it is also called the 3.
5th generation or fourth generation sequencing technology
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There are many specific product types of Nanopore sequencers, all of which are platforms based on Nanopore chips, ranging from the PromehION and GridION series sequencers composed of multiple chip arrays to the MnION that can be connected to the Type C interface of mobile phones and the USB interface of computers.
Series Portable Sequencers
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Among them, PromethION is a high-throughput, high-sample-count benchtop system with 48 sequencing chips, each with up to 3,000 nanopore channels, for a total of 144,000 nanopore channels
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Based on this modular design, flow cells can be run individually or simultaneously, which is especially suitable for projects with large sample sizes and huge data volumes
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Both of the above two types of third-generation sequencing platforms have the advantages of long read length, no GC bias, and direct detection of methylation modifications
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In comparison, the SMRT platform has no error preference, and can improve the sequencing accuracy by increasing data error correction; the Nanopore platform has longer read lengths, which can reach the Mb level, and the MnION sequencer is the size of a mobile phone and is relatively portable (see details for details).
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The application direction remains to be explored.
At present, the third-generation sequencing platform of long-read sequencing has been widely used in scientific research projects in the fields of complex animal and plant genomes, microbial genomes, full-length transcriptomes, microbial population research, and human genome variation detection.
The bottleneck problem of detection technology in the above fields
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In terms of disease detection, third-generation sequencing technology is based on the characteristics of single-molecule detection and long-read sequencing.
Methylation detection has unique advantages
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Although many genetic diagnostic products based on second-generation sequencing technology have basically matured, due to the limitations of high-throughput sequencing technology, such as short read length and uneven coverage of the genome, the detection of SNP and InDels is acceptable, but the detection of complex structural variants is acceptable.
detection is ineffective
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The third-generation sequencing technology has the advantages of long read length, no PCR amplification, and no GC preference.
Detecting structural variants such as deletions, duplications, inversions, and translocations, which further improves the detection rate of diseases and effectively makes up for the lack of structural variant detection by second-generation sequencing technology
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Compared with the second-generation sequencing platform, the third-generation sequencing platform has greatly improved the sequence read length.
However, due to its high error rate, cost, and sample requirements, the supporting technologies such as algorithms, software, and databases still need further research.
The platform is still in the application stage of scientific research projects
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In response to the above problems, various platforms are constantly optimizing and upgrading related technologies.
For example, the HIFI technology mode of the PacBio sequencer can effectively improve the data accuracy, and the Nanopore platform has also upgraded the PromethION to further improve the detection accuracy
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It is believed that with the continuous development of the third-generation sequencing platform, after its translational application in the medical field in the future, it will effectively fill the gap of the current detection methods for related diseases such as genome structural variation
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(Author: Center for Medical Device Technology Evaluation, State Drug Administration)