高通量测序技术

2003年4月人类基因组计划完成，开启了一个全新的时代：后基因组时代。也许需要很久的时间，人类才能完全明白这意味着什么。随着科普工作不断向前地进行，高通量测序技术（NGS）已经应用于生命科学的方方面面。
The completion of the Human Genome Project in April 2003 ushered in a new era: the post-genomic era. It may be a long time before humanity fully understands what this means. With the advancement of science popularization, high-throughput sequencing technology (NGS) has been applied to all aspects of life science。

测序技术是基因组学的核心技术。其实，测序技术的发展主要基于两个非常具有里程碑意义的理念：“生命是序列的”和“生命是数据的”。序列是基因组学最基本最重要的数据，也是生命科学领域大数据时代的核心组成部分。简单来说，测序技术就是将DNA/RNA分子中碱基ATGC的排列顺序显示出来。
Sequencing technology is the core technology of genomics. In fact, the development of sequencing technology is based on two landmark ideas: "Life is sequence" and "life is data". sequence is the most basic and important data in genomics, as well as the core component of the big data era in the field of life science. In simple terms, sequencing technology is to display the sequence of the ATGCs of the bases in a DNA/RNA molecule.
1953年，Watson和Crick提出DNA双螺旋结构
In 1953, Watson and Crick proposed the double helix structure of DNA.

DNA双螺旋模型以及“生命是序列的”观点的发表，直接推动了测序技术的发展，因为解读生命遗传信息的前提就是得到它的载体——序列。
从20世纪70年代到现在有很多测序技术和平台的产生，其中包括SBC法、454、Ion Torrent、SBL法、Sanger法、Illumina、Pacbio、Nanopore等。
The publication of the DNA double helix model and the view that "life is a sequence" directly promoted the development of sequencing technology, because the premise of interpreting the genetic information of life is to obtain its carrier - sequence.
Since the 1970s, many sequencing technologies and platforms have been produced, including SBC method, 454, Ion Torrent, SBL method, Sanger method, Illumina, Pacbio, Nanopore and so on.
一个以NGS为核心的完整产业链已经形成了
A complete industrial chain with NGS as the core has been formed

第一代测序技术
First generation sequencing technology
Sanger法是基于DNA合成反应的测序技术，又称为SBS法、末端终止法。1975年由Sanger提出，并于1977发表第一个完整的生物体基因组序列。
核心原理：由于双脱氧核苷酸(ddNTP)的3’位置脱氧，其在DNA的合成过程中不能形成磷酸二酯键，因此可以用来中断DNA合成反应，在4个DNA合成反应体系中分别加入一定比例带有放射性同位素标记的ddNTP（分为：ddATP,ddCTP,ddGTP和ddTTP），通过凝胶电泳和放射自显影，根据电泳带的位置确定待测分子的DNA序列。
在每个反应体系中，ddNTP相对于dNTP是很少的，所以只有部分新链在不同的位置特异性终止，最终就会得到一系列长度不一的序列。
Sanger method is a sequencing technology based on DNA synthesis reaction, also known as SBS method and terminal termination method.
It was proposed by Sanger in 1975 and the first complete genome sequence of an organism was published in 1977.
Core principle: Dideoxy nucleotide (ddNTP) can not form phosphodiester bond in the process of DNA synthesis due to its deoxygenation at the 3 'position, so it can be used to interrupt the DNA synthesis reaction. A certain proportion of radioisotopically labeled ddNTP is added to the four DNA synthesis reaction systems (divided into:ddATP,ddCTP,ddGTP and ddTTP), and the DNA sequence of the molecule is to be tested was determined by gel electrophoresis and autoradiography according to the position of the electrophoretic band.
In each reaction system, ddNTP is rare relative to dNTP, so only some of the new strands terminate specifically at different positions, resulting in a series of sequences of varying length.

第二代测序技术
Second generation sequencing technology
以Illumina平台为代表的第二代测序技术实现了高通量测序，有了革命性进展，使得大规模并行测序成为现实，极大推动了生命科学领域基因组学的发展。Illumina循环SBS法(cycle SBS)即SBRT(Sequencing By Reversible Termination,可逆终止)的核心技术是DNA合成的可逆性末端循环，即3'-OH可逆性的修饰和去修饰。
基本原理：将dNTP的3'-OH以叠氮集团RTG(Reversible Terminating Group,可逆末端基团)进行修饰；将4种碱基分别与不同的荧光分子连接；DNA合成时，RTG能起到类似于ddNTP的作用终止反应；每次合成反应终止并读取信号之后，洗脱RTG和荧光分子，进行下一轮循环。
Second generation sequencing technology, represented by Illumina platform, has achieved high-throughput sequencing and made revolutionary progress, making massively parallel sequencing a reality and greatly promoting the development of genomics in the field of life sciences.
The core technology of Illumina (Cycle SBS）,SBRT(Sequencing By Reversible Termination) is Reversible end-cycle of DNA synthesis, namely modification and de-modification of 3'-OH reversibility.
Basic principle: 3'-OH of dNTP was modified in Reversible Terminating Group (RTG);
The four bases were connected to different fluorescent molecules.
During DNA synthesis, RTG plays a similar role to ddNTP to terminate the reaction.
After each synthesis reaction is terminated and the signal is read, the RTG and fluorescent molecules are eluted for the next cycle.
主要过程：
The main process
a, DNA待测文库构建
利用超声波把待测的DNA样本打断成小片段，并在这些小片段的两端添加上不同的接头，构建出单链DNA文库。这些文库中的DNA在通过flowcell(吸附流动DNA片段的槽道)时会随机附着在flowcell表面的channel上。每个Flowcell有8个channel，每个channel的表面都附有很多接头，这些接头能和建库过程中加在DNA片段两端的接头相互配对，并能支持DNA在其表面进行桥式PCR的扩增。
a, DNA library construction
Single-stranded DNA libraries are constructed by using ultrasound to break the DNA sample under test into small fragments and adding different joints at each end of these fragments.
DNA from these libraries will randomly attach to channels on the surface of Flowcell when passing through Flowcell (channels that adsorb flowing DNA fragments).
Each Flowcell has 8 channels, and the surface of each channel is attached with many joints. These joints can be paired with the joints added at both ends of the DNA fragment during library construction and can support DNA amplification by bridge PCR on its surface.

b,桥式PCR以Flowcell表面所固定的接头为模板，进行桥形扩增。经过不断的扩增和变性循环，最终每个DNA片段都将在各自的位置上集中成束，每一个束都含有单个DNA模板的很多份拷贝，进行这一过程的目的在于实现将碱基信号强度放大，以达到测序所需的信号要求。
b. Bridge PCR was performed using the fixed connector on the Flowcell surface as template for bridge amplification.After repeated cycles of amplification and denaturation, each DNA fragment will eventually be assembled into bundles at its location, each containing many copies of a single DNA template. The purpose of this process is to amplify the strength of the base signal to meet the signal requirements for sequencing.

测序方法边合成边测序：
Synthesize and sequence as they go

1. dNTP模型
   dNTP model
   
2. dNTP加上可终止反应的基团RTG和荧光信号
   dNTP add the group RTG and fluorescence signal that can terminate the reaction
   
3. 反应体系中同时添加DNA聚合酶、接头引物和带有碱基特异荧光标记的4种dNTP
   The reaction system was supplemented with DNA polymerase, adaptor primer and four kinds of dNTP with base-specific fluorescent labeling
   
4. 脱掉-OH和二磷酸，进行合成
   Take off the OH and the diphosphate, and synthesize it
   
5. 反应因RTG终止，激发荧光进行信号采集
   The reaction was terminated by RTG, and fluorescence was excited for signal acquisition.
   
6. 洗脱RTG和荧光分子，进行下一轮循环
   The RTG and fluorescent molecules were eluted for the next cycle.
   
   第三代测序技术
   Third generation sequencing technology

以Pacbio平台为代表的SMRT(Single-Molecule Real Time Sequencing,单分子实时测序)测序技术具有高通量、长读长的特点。
Single-molecule Real Time Sequencing (SMRT) technology represented by Pacbio platform has the characteristics of high throughput and long read.
基本原理：Pacbio仍然采用边合成边测序的原理，但实现了两个重要的技术突破。一个是将荧光分子标记在磷酸上，这样在反应停止且捕获荧光信号以后，可直接随磷酸基团脱落，解决了因噪音污染导致的读长很短的问题；二是由于不需要PCR扩增，信号的有效提取成为了关键。通过引入零模波导孔（ZMW）技术解决这一问题。在纳米室底部有一个孔径70nm的小孔，由于远远小于激光的波长，所以激光从底部照射时，只会照亮一个小的区域，提高了信噪比。
The rationale: Pacbio still uses the principle of synthesis-as-you-go sequencing, but achieves two important technological breakthroughs.
One is to mark the fluorescent molecules on the phosphoric acid, so that after the reaction stops and the fluorescence signal is captured, it can be directly shed with the phosphoric acid group, which solves the problem of short read length caused by noise pollution.
Second, because no PCR amplification is required, effective signal extraction becomes critical.
Zero mode waveguide hole (ZMW) technology is introduced to solve this problem.
At the bottom of the nanochamber, there is a small hole with a 70nm aperture, which is much smaller than the wavelength of the laser, so when the laser shines from the bottom, it will only illuminate a small area, improving the signal-to-noise ratio.

主要过程：如下图所示，类似于Illumina部分展示的模式图，也是边合成边测序。
Main process: The following diagram is similar to the pattern shown in the Illumina section, which is also synthetically sequenced.

第四代测序技术
Fourth generation sequencing technology
纳米孔测序技术是单分子实时测序的新一代技术，主要是通过ssDNA或RNA模板分子通过纳米孔而带来的“电信号”变化推测碱基组成进行实时测序。
Nanopore sequencing technology is a new generation of single-molecule real-time sequencing technology, which is mainly based on the "electrical signal" change of ssDNA or RNA template molecules through the nanopore to infer the base composition of real-time sequencing.

基本原理：当纳米孔充满导电液时，两端加上一定电压，分子模板通过纳米孔生成可测量电流。纳米孔的直径只能容纳一个核苷酸，单链模板就会在电场作用下依次通过纳米孔而引起电流强度变化，通过检测相应的电流峰判断碱基，实现实时测序。
Basic principle: When a nanopore is filled with a conductive fluid, a voltage is applied to both ends, and the molecular template generates a measurable current through the nanopore.
The diameter of the nanopore can only accommodate one nucleotide, and the single-stranded template will pass through the nanopore in turn under the action of electric field, resulting in the change of current intensity. The base can be determined by detecting the corresponding current peak to realize real-time sequencing.

四大测序技术的优缺点：

Sanger法测序读长长、准确度高，但是通量不高；

Illumina测序读长短、通量高、准确度高，在进行基因组组装或者结构变异分析的时候没有优势，可用作三四代测序read的纠错；

Pacbio测序读长长、通量高、准确度不高，但可通过测序深度弥补，GC偏差低，可进行甲基化的直接测序。

Nanopore测序读长长、通量高、准确度低，不可通过测序深度弥补，但可通过Illumina read 纠错。

第一、二、三代测序技术都是基于边合成边测序的原理，因此Nanopore技术被一些人称为第四代测序技术。
NGS是一个朝阳产业，它给我们带来不同层次的惊喜，如单细胞技术和空间组学。所以这一行业的从业者往往从最前言的学术文章，技术文档，项目执行中学习这方面的知识。
Advantages and disadvantages of the four sequencing technologies:
Sanger sequencing has long read length and high accuracy, but low throughput.
Illumina sequencing has high read length, high throughput and high accuracy, which has no advantages in genome assembly or structural variation analysis. It can be used as the error correction of third and fourth generation sequencing READ.
Pacbio sequencing has long read length, high throughput and low accuracy, but it can be compensated by sequencing depth. It has low GC deviation and can be used for direct methylation sequencing.
Nanopore sequencing has long read length, high throughput and low accuracy, which cannot be remedied by sequencing depth, but can be corrected by Illumina read.
First -, second -, and third-generation sequencing technologies are all based on the principle of synthesis-on-the-go sequencing, so Nanopore technology is called fourth-generation sequencing by some people.
NGS is a sunrise industry, which brings us different levels of surprise, such as single cell technology and spatial omics.
So people in this field tend to learn from the best academic articles, technical documentation, and project execution.

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中国科学技术大学20220916