Knife out of the sheath: How does single-cell sequencing technology fight disease?

From Mendel's discovery of the secret that genes determine biological traits in his own pea fields, to the later discovery that nucleic acid sequences can encode life, to biological cloning and gene editing, the development of genetic technology has gradually made the blueprint of life become clear

We all develop from the combination of sperm and egg, but the cells of our body are very different: some cells bear power, some cells protect the body, and although they share the same DNA, they exhibit different shapes and functions.

All this stems from the complexity of the differential expression of genes themselves in different cells

As a tool that can efficiently detect the heterogeneous expression of genes in different cells, single-cell sequencing has been pushed to the forefront of the times and has become an indispensable and powerful technology in the field of life medicine

Why single-cell RNA sequencing?

The characteristics of a cell are determined by its DNA

How to extract the cancer cells hidden in the body has become a major medical problem

The difference between cancer cells and other normal cells is very small.

Therefore, it is necessary to check the cells one by one for cancer treatment

Direct protein analysis of cells is difficult

mRNA is the messenger of gene expression.

In addition to tumors, single-cell RNA sequencing technology can also detect the mRNA expression profiles of different cells in embryonic development stages, so as to screen out key genes at different organ development stages and provide a reference for stem cell organ transplantation

In addition, we can also directly sequence the common DNA mutation sites in cancer cells to analyze whether these key DNA sequences have potential mutation risks, so as to judge the condition of this cell

At present, single-cell sequencing has been used in a series of biomedical research and clinical trials such as tumor detection, embryonic development, immune cell therapy and stem cell differentiation, and has made a very important contribution to human beings to explore the secrets of life and fight diseases

How to achieve single-cell sequencing?

The steps of single-cell sequencing are very simple, requiring only three steps: nucleic acid extraction, sequencing, and sequencing library construction

For single cell sequencing, we only need to separate each cell, and then sequence the cells individually

Analyze the sequencing ideas one by one: separate individual cells by techniques such as flow cell sorting or laser capture microdissection, and then perform nucleic acid extraction

The clever application of barcode improves the efficiency of single cell identification: we don't need to separate and extract individual cells one by one, only need to add a unique nucleic acid sequence tag to each cell and then sequence analysis
.

This strategy is like a bank's number queuing system: different customers are marked with different marks, and specific windows are assigned to queue on demand
.
Numerous "lobby manager" hydrogels handed different "nucleic acid tag" numbers to different customers, and took them one by one into the hall to process "sequencing" business
.
In one experiment, hundreds of thousands of single cells can be measured
.

In addition, it is necessary to verify the results of single-cell sequencing.
Commonly used methods are Bulk (mixed pool) sequencing and proteomics analysis
.
Bulk sequencing directly sequence the entire tissue, which is complementary to single-cell sequencing
.
Bulk sequencing is like a comprehensive fruit and vegetable juice squeezed from all the fruits together.
It can be compared with the results of single-cell sequencing to determine the relationship between the single-cell sequencing results and the whole, and then accurately define the "abnormal"
.
Proteomics directly analyzes the expression of key proteins in cell populations, which echoes mRNA sequencing
.

How is DNA sequenced?

The earliest gene sequencing technology is based on the process of DNA replication, which converts DNA sequence information into readable signals
.

The process of gene replication is like splicing Lego blocks: using an original DNA strand as a template, and according to base complementary pairing, DNA synthetase splices nucleotides together one by one
.

First-generation sequencing (Sanger method) uses DNA polymerase synthesis reaction (PCR) to incorporate a small amount of radioisotope-labeled ddNTP into the normal PCR reaction system to determine a complete DNA sequence
.

The special structure of ddNTP will terminate the PCR reaction.
Like a Lego brick without protrusions, the following bricks can no longer be linked, so the bricks cannot be extended
.
Since the position of ddNTP in the entire PCR reaction is random, the PCR reaction can be terminated at any position.
If we let all the positions of the base appear, there will be at least one synthesis termination, and each termination position is the base.
The position of appearance; Finally, through gel electrophoresis and autoradiography imaging, we can get the base information of all the stop sites, thereby obtaining a complete DNA sequence
.

First-generation sequencing is the simplest and has high accuracy, but it can only measure a limited length at a time
.
Just like playing Snakes, the longer the snake becomes, the more difficult it is to control.
The longer the sequence is, the more unstable it becomes
.
The longest one-generation sequencing can measure the sequence of about 1000 bp, and there is no way to start the longer sequencing, and there is no way to deal with the whole genome of hundreds of millions of bases
.

Since we can't measure the entire sequence at once, we can measure a part of the genome and splice it together!

Next Generation Sequencing (NGS) is based on the "shotgun method" to break up the lengthy genome, sequence the fragments separately, and finally splice them into a complete sequence
.
Since the detailed DNA library of common species has been constructed, in diagnosis and treatment applications, we only need to compare a small piece of DNA with the database to find the original DNA in this library
.
The whole process is like searching, instead of looking for pieces of related sequence fragments like a puzzle
.

Although the second-generation sequencing is still based on the DNA amplification process of PCR, it uses the principle of “sequencing while synthesizing” to allow the four bases to generate their own specific fluorescent signals during DNA synthesis, which are simultaneously captured by a precision optical camera and directly Read base information instantly
.

Second-generation sequencing has greatly improved the throughput of sequencing, reduced the cost and cycle of sequencing, and has a high degree of automation.
It has become the most eye-catching sequencing technology on the market today
.

Second-generation sequencing still has its own shortcomings: the fluorescence of a single base is very weak, and optical instrument reading is prone to errors; sequencing fragments need to be replicated and amplified, and the replication process will occasionally cause base mismatch and loss.
These errors will be caused by sequencing.
The sample size increases and accumulates; the length of a single sequence is small, and it is prone to mismatches when compared with the database
.

Because of these technical shortcomings, people are eager to come up with technologies that do not need to be expanded and read longer
.

While retaining the high-throughput characteristics of second-generation sequencing, third-generation sequencing uses single-molecule reading technology to eliminate the need for the amplification process in PCR, which effectively reduces the loss and mismatch of base information due to amplification.
, And increased the sequencing length to about 10kb
.

At present, the second and third-generation sequencing technologies are all based on capturing weak fluorescent signals, so they need to be equipped with expensive optical monitoring systems; based on the principle of DNA replication, they rely too much on the activity of DNA polymerase.
These features greatly increase the efficiency of sequencing.
Cost
.

The fourth-generation sequencing technology attempts to complete sequencing without the need for DNA synthesis
.

Fourth-generation sequencing uses primers to pass the sequence to be tested through a nanopore, and reads the difference in electrical signals when different bases pass through the nanopore for real-time sequencing
.
This technology not only reduces the cost of the instrument and consumables, but also achieves excellent accuracy.
It can also reduce the mechanical volume and make a sequencing instrument the size of a desktop computer the size of a U disk
.
The first domestic fourth-generation sequencer developed by QC Technology provides technical support for a large-scale clinical application of single-cell sequencing
.

The application and future of single-cell RNA sequencing

In 2009, mRNA-Seq, the first single-cell transcriptome sequencing technology, opened the history of single-cell sequencing in the biomedical field
.
In 2014, the single-cell sequencing technology was rated as the technology of the year by the top international academic journal "Nature-Methods"
.
In the same year, "Science" magazine ranked single-cell sequencing as the most noteworthy field of the year, believing that this technology has brought unprecedented changes to the biological and medical communities
.

In 2017, a "Human Cell Atlas Project" comparable to the "Human Genome Project" was born
.
The "Human Cell Atlas Project" is an international large-scale collaborative project whose purpose is to classify and define the functions of all human cells based on unique biological macromolecular information
.
An extremely critical technology in this project is single-cell RNA sequencing
.
Single-cell RNA sequencing can reveal the gene structure and gene expression status of a single cell, reflecting the differences between cells, and its application in the field of life medicine has become more and more extensive
.

In the near future, single-cell sequencing technology can push precision medicine to a broader space, and more patients can enjoy affordable specific and efficient therapies, and improve treatment efficiency and experience in the treatment process
.

Single-cell RNA sequencing has become an indispensable technology in the field of life medicine
.
Now it plays an important role in tumor, developmental biology, microbiology, neuroscience and other fields
.
With the continuous improvement and mutation of technology, single-cell sequencing technology will have a very broad application prospect in the precise research and treatment of diseases in the future, and it will shine more brilliantly on the stage of human health
.

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