What are the types of single-cell sequencing?

Sequencing nucleic acid molecules 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.

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Types of single-cell sequencing

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

• Single-cell genome sequencing

• Single-cell transcriptome sequencing

• Single-cell methylome sequencing

Single-cell genome sequencing

Single-cell genome sequencing comprises isolating a single cell, processing and amplifying the whole genome, constructing the sequence libraries, and applying next-generation sequencing techniques. It is also called genome sequencing.

Methods

There are over a hundred methods available for this. These include the following:

• Single-cell DNA template strand sequencing (Strand-seq): This method helps discover genomic structural variations. It enables the discovery of a full spectrum of structural variation classes on a somaticFocused on a bodily or physical form level. These variations are $\ge 200$ kilobytes in size.

• Multiple displacement amplification (MDA): This widely-used method allows the amplification of different weights of the DNA. It requires reagents: DNA polymerase, and random primers for isothermal amplificationA method to detect a nucleic acid target sequence. MDA produces results of $12$ kilobytes to$100$ kilobytes in length.

• Multiple annealing and looping-based amplification cycles (MALBAC): This method is similar to MDA, but the primers are covered with a common sequence for the downstream polymerase chain reaction (PCR). This common sequence enables self-litigationThis is the process of tying a ligature around a blood vessel or some duct. It refers to DNA sequences, here. and the formation of loops to prevent more applications. These loops are denatured in successive temperature cycles, allowing the fragments to amplify due to PCR.

Applications

Single-cell genome sequencing has the following applications:

• It helps obtain microbial genome sequences without cultivation.

• It is used in cancer sequencing.

Single-cell transcriptome sequencing

Single-cell transcriptome sequencing (scRNA-seq) helps us express profiles of singular cells. It is considered one of the best methods for describing phenotypes and cell states.

Methods

This method involves isolating cells and their RNA, the reverse transcriptionThe process of generating cDNA from RNA templates of the cells, amplification, library generation, and sequencing.

Recent progress in this sequencing allows the encapsulation of individual cells in a microfluidic device. Here, the RNA is converted to cDNAComplementary DNA by reverse transcription. Each droplet carries a unique barcode for labeling each cDNA derived from a single cell. After this, we can mix cDNAs from various cells for sequencing.

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

Applications

Single-cell transcriptome sequencing has the following applications:

• Data from this sequencing method is used to increase the signal-to-noise ratio in machine learning models.

• It helps determine heterogeneity across a population.

• It helps in cluster analyses.

• It provides insight for the development of organisms.

• It is used across neurology, oncology, immunology, and infectious diseases, and cardiovascular research.

Single-cell methylome sequencing

Single-cell methylome sequencing is based on the methylationThe biological process in which methyl groups are attached to the DNA of the DNA. The few known types of methylation that occur in nature include the following:

• 4mC 4-methylcytosine

• 6-methyladenine

• 5-hydroxymethylcytosine

• 5-methylcytosine

Methods

There are two methods for single-cell methylone sequencing. These are as follows:

• Single-cell reduced representation bisulfite sequencing (scRRBS): This method uses one or multiple restriction enzymes on the DNA. This produces sequence-specific fragmentation of the DNA. We then treat this fragmented DNA with bisulfite, and sequence it. This method tends methylated cytosines to cluster at CpG islandsThe markers used in organisms that have 5-methylcytosine in their genomes.

• Bisulfite sequencing: Bisulfite sequencing is a primary method of sequencing 5-methylcytosine using singular cells. This method requires the treatment of the DNA with bisulfite. This converts the obtained cytosine residues into uracil, but leaves behind the 5-methylcytosine unaffected. Then, the treated sequence is aligned to an unmodified genome to obtain a readout. We add adapters after the bisulfite fragmentation process is done on the DNA. This allows the amplification of the fragments due to PCR.

Applications

Single-cell methylome sequencing has the following applications:

• It helps us explore the epigenetic differences in similar cells.

• It helps us identify distinct cell types by hierarchal classification.

• It helps the emergence of different cell types from a singular embryo.

• It helps us study rare but active cell types in cancer.