What is single-cell sequencing?

Sequencing nucleic acid moleculesMolecules like the DNA and RNA provides us with the genetic instructions required for life. This sequencing now enables us to sequence the human genome.

The concept of sequencing started in the 1970s. It stems from the works of Walter Fiers, Ray Wu, and Frederick Sanger. Commercial DNA sequencers were produced in the 1990s and called the next generation sequencers (NGS).

Single-cell sequencing was one of the breakthroughs of these sequencing methods.

Single-cell sequencing

Single-cell sequencing is the sequencing of individual cells to obtain genomic, multi-omic, and transcriptomic information at a single cell level.

We currently use single-cell sequencing to measure each population cell's genome (scDNASingle cell DNA-seq), DNA-methylome, and transcriptome (scRNASingle cell RNA-seq).

This technique examines the information acquired from singular cells with next-generation sequencing technology. This results in a higher resolution of cellular differences, and a better understanding of a singular cell's function in the context of its microenvironment.

How to perform single-cell sequencing

Single-cell sequencing technologies work in four significant steps. These are as follows:

1. We isolate the single cells from a cell population.

2. We extract, process, and amplify the genetic material of each isolated cell.

3. We prepare the "sequencing library" that includes the genetic material of an isolated cell.

4. We sequence the library through next-generation sequencing.

Let's discuss these steps in detail:

Step 1: Isolating the cells

We can isolate cells using different methods. The technique depends on the nature of the sample and the processing steps after the isolation.

The following factors of the technique define its performance:

• Recovery: This is the portion of the target cells collected compared to the total number of cells initially available.

• Purity: This is the part of the target cells collected.

• Efficiency: This is the number of cells that can be isolated per unit of time.

Some of the commonly used techniques are as follows:

• Manual cell picking

• Laser capture microdissection

• Magnetic-activated cell sorting

• Fluorescence-activated cell sorting

Step 2: Extracting, processing, and amplifying

To check the quality of cell isolation, we evaluate the RNA integrity for the scRNA-seq analyses and lyseDisintegrating a cell by rupturing the cell membrane the isolated cellsThe disintegration of a cell by rupture of the cell membrane..

We isolate and amplify the genetic material's parts of interest to provide enough material for subsequent detection. We do this because single cells usually yield tiny amounts of DNA or RNA. The result of this is single-stranded DNA.

For the amplification step, we usually use a polymerase chain reaction (PCR) or in vitro transcription (IVT).

Step 3: Preparing a sequencing library

We turn the amplified DNA into a sequencing library before it is sequenced. A sequencing library is a group of single-stranded DNA parts derived from one cell population or one specific cell.

We uniquely barcode the DNA parts to identify the initial cell they belong to, and add adapter sequences to the 5' and 3' ends. The DNA is now called an insert. The barcodes and adapters cap the insert from both ends.

We barcode DNAs with similar sequencing libraries using identical oligonucleotide sequences. This allows the amalgam of different libraries to be sequenced together at run time.

Kits are available for the sampling and library preparation steps.

Step 4: Sequencing

There are many sequencing technologies available. We focus on sequencing by the synthesis method.

After the amplification step, the DNA fragments clone. Each set of clones emits identical signals during the sequencing process. The resulting clusters are strong enough for detection. This sequencing usually takes place on chips.

We place the adapters and other required materials on the chips, where they interact with the inserts. The insert sequencing has to replicate multiple times using a polymerase and fluorescently tagged nucleotides.

In each cycle, we add a fluorescently tagged nucleotide. If incorporated by polymerase, light emission is triggered for each specific nucleotide. For each cycle, we capture the light spectra. Each nucleotide emits a different light; cycle by cycle, the sequencer reconstructs the sequence of all inserts. It also reads the insert's tags to assign each measurement to the allotted library.

Note: Other methods for this sequencing include ligation, and proton detection.

Types of single-cell sequencing

There are different types of single-cell sequencing based on what they measure. These are as follows:

• Single-cell genome sequencing

• Single-cell transcriptome sequencing

• Single-cell methylome sequencing

Note: To learn about these sequencing types, we can visit here.

Conclusion

Single-cell sequencing is essential as it has various advantages. Some of its impacts include the following:

• It lets us probe each cell and measure its specific contribution to the whole cell population, organism, or population.

• It lets us study rare cells.

• It lets us explore similar phenotype variations within the same cell type populations.

• It lets us investigate rare antigen-specific cells.

• It lets us measure the structure and composition of microbes.

• It lets us discover unknown gene functions.

• It lets us study tumor progression mechanisms.

Other than these, it has many more applications in different fields based on extracted cells.