2.2 Single-cell transcriptome sequencing
Single-cell transcriptome sequencing is commonly referred to as single-cell RNA sequencing (scRNA-seq). It is used to analyze single-cell-level cell type differences and gene expression profiles in complex biological systems. It is defining new cell types and discovering new biomarkers. It also has unique advantages in analyzing and understanding cellular heterogeneity. Single-cell RNA sequencing is easier to perform than single-cell DNA sequencing because each cell contains many RNA molecules, but the number of DNA molecule copies is extremely small. When performing routine gene expression analysis, the 3'end of the gene is generally selected for sequencing, and when conducting immune cloning and related studies of T cell receptor (TCR) and B cell receptor (BCR), the 5'end of the gene should be selected for sequencing. Sequencing at the 5'end will assemble and annotate the full-length V(D)J fragments, match the alpha and beta chain sequences of a single T cell TCR, and match the BCR light and heavy chain sequences of a single B cell. It is of great significance for exploring the adaptive immune system.

2.3 Single cell DNA methylation sequencing
During normal cell development, DNA methylation is an important epigenetic marker in many species, and it exists as a chemical or protein tag in the DNA sequence. Without changing the existing DNA sequence, this tag tracks the developmental changes of the cell, participates in the decision to turn on or off the biological control process, and controls the fate of the cell. Previous research on DNA methylation was based on the overall measurement of a large number of cells, while ignoring the differences between cells and the effects of some rare cell types. Therefore, the ability to measure DNA methylation in a single cell may make an important contribution to understanding several key biological processes such as embryonic development, disease progression, and aging.

2.4 Single cell histone modification sequencing
Histone modifications can modulate the affinity and accessibility of certain DNA regions. In order to analyze the role of histone modifications in different cells, four methods include single-cell chromatin immunoprecipitation sequencing (scChIP-seq), single-cell chromatin integration labeling followed by sequencing (scChIL-seq), single-cell cleavage undertargets and tagmentation ( scCUT&Tag) and single-cell chromatinimmune-cleavage followed by sequencing (scChIC-seq) need to be used. These advanced single-cell technologies can reveal various aspects of epigenetic heterogeneity in cases where cells cannot be analyzed by transcriptome alone and are expected to find more applications in cancer genomics and related transformation studies.

2.5 Single cell chromatin structure sequencing
The chromatin structure plays a central role in embryonic development, differentiation and disease development. Single-cell sequencing for transposase accessible chromatin (scATAC-seq) is a solution for functionally identifying relevant changes in chromatin structure in cells. Using scATAC-seq allows researchers to identify the variability of the genomic location of open chromatin sites in each cell, thereby gaining insight into the intercellular variability produced by other identical DNA sequences. Using scATAC-seq can also enable researchers to observe that chromatin densification and DNA binding proteins can precisely regulate gene expression to achieve the purpose of complementing DNA copy number variation and RNA expression data obtained from single-cell DNA and RNA sequencing. Furthermore, functional identification of relevant changes in chromatin structure in specific subsets of cancer cells is a unique advantage of scATAC-seq.

2.6 Single-cell spatial transcriptome sequencing
In an environment where single-cell sequencing technology is becoming more and more mature, single-cell transcriptome sequencing technology based on single-cell transcriptome sequencing has emerged, which combines Visium spatial endoscopy technology with single-cell transcriptome sequencing to produce a Visium Spatial gene expression solution. Using this scheme, the total mRNA in the tissue section can be measured, and only the Visium Spatial slide with the complete tissue section is required as input, and the gene expression in the cell corresponds to the tissue section without deviation. However, in order to ensure the integrity of mRNA transcripts and maintain the morphological quality of tissue sections, correct tissue processing and preparation techniques are extremely important for this scheme. The emergence of this method has made it possible to explore the transcriptome at the single cell level in tissues that were difficult to dissociate in the past and some rare cell types that are highly prone to stress death.

3 Application of sc RNA-seq
3.1 Application of sc RNA-seq in the field of cancer research
The heterogeneity of cancer comes from the diversity of clones and the evolution of mutations, which promotes the metastasis of cancer cells and discovers the diagnosis and treatment of cancer. As an ideal tool, SCS has been increasingly used to reveal the heterogeneity in various primary tumors, such as breast cancer, lung cancer, colon cancer and so on.
Using single-cell resolution to study the dynamics of genomic cloning in breast cancer patients suggests that genomic aberrations may be a repeatable determinant of evolutionary trajectories. Interestingly, when studying colon cancer, single cell whole genome sequencing revealed a large number of mutant genes SLC12A5 at the individual level, and found that colon cancer cells has dual origin but then developed into 2 different tumor cell subpopulations. However, another study using a single cell exon sequence revealed the evolution of bladder cancer. The results of this study showed that 66 individual bladder cancer cells were formed by a single cell division, but then developed into 2 different tumor cell subunits. The clone structure can be determined by analyzing a large number of single cell samples extracted from the cells of patients with three types of myelodysplastic syndrome (MDS). Single-cell exogenous gene sequencing revealed the monoclonal evolution that occurred in JAK2-negative bone marrow boosting tumors, and further identified mutations in tumor candidate genes.
In recent years, a series of studies have used SCS to understand different rare circulating tumor cells (CTCs). CTCs are a type of cancer cells that shed from the primary tumor and enter the peripheral blood of patients. They are rare in number, but are closely related to the metastasis and recurrence of tumor cells. There are already many platforms for detecting and enriching CTCs. Through SMART-seq analysis of tumor cells in a single circulatory system, different expressions of multiple genes were found, and most active genes were detected in prostate lymph node cancer cells. At the same time, different gene expression patterns were also found. The number of CTCs has been used as a prognostic indicator to predict the survival rate of cancer patients. However, CTCs should have greater potential. The study of CTCs at the single cell level may help to uncover the underlying mechanisms of tumorigenesis and metastasis. Although this field has not yet developed, it still has great potential in providing new clinical applications and in-depth understanding of tumor metastasis and cancer development.
To be continued in Part III…

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