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Single cell sequencing:related concepts and development process

Recently, we sorted out the sequencing part of molecular diagnosis technology. The sequencing technology includes DNA sequencing, RNA sequencing, single cell sequencing, methylation sequencing, etc. according to the different types of samples. At the beginning of this issue, we will introduce single-cell sequencing technology one by one from the following aspects:the concept and development of single-cell sequencing technology, the operation process of single-cell sequencing technology, single-cell whole genome sequencing technology, single-cell whole transcriptome sequencing technology, and the application of single-cell sequencing technology.

Single cell sequencing technology

Single cell sequencing (SCS) technology refers to the amplification and sequencing of transcriptome or genome at the single cell level to detect the data of single cells in genomics, transcriptomics, epigenomics and proteomics. It mainly involves single-cell genome sequencing, single-cell transcriptome sequencing and single-cell epigenome sequencing.

Single cell genome sequencing (Fig. 1a):it is used to amplify a small amount of whole genome DNA of isolated single cells, obtain a complete genome with high coverage, and then conduct high-throughput sequencing, which is used to reveal genetic variations in single cells, such as single nucleotide variation (snvs), copy number variation (CNVs) and genome structure variation (SVS), cell population differences and cell evolution.

Single cell transcriptome sequencing (Figure 1b):it is to amplify the micro full transcriptome RNA of isolated single cells and then conduct high-throughput sequencing, which is used to generate maps of gene expression, gene fusion and alternative splicing in single cells. This technology is considered to be the gold standard for defining cell status and phenotype by 2020. [1]

Single cell epigenome sequencing (Figure 1c):it is to detect the heritable changes of phenotype under the condition of unchanged DNA sequence, including DNA methylation, histone modification, chromatin accessibility, etc. In eukaryotes, 5-methylcytosine (5mC) is widely distributed in the genome and plays an important role in regulating gene expression by inhibiting transposable factors [2]. Sequencing 5mC in a single cell can reveal how epigenetic changes in genetically identical cells from a single tissue or population produce cells with different phenotypes. Single cell bisulfite sequencing is the gold standard for DNA methylation research.

图1 Single cell sequencing technology应用范围示意图[3]

A:Application scope of single cell genome sequencing; B:Application scope of single cell transcriptome sequencing; C:Application scope of single cell DNA methylation sequencing;

Why single cell sequencing?

Multicellular organisms will inevitably bring differences between different cells in the process of cell division and differentiation, forming the heterogeneity of genetic information. The information obtained by traditional detection methods comes from the mixed samples of millions or even more cells, so the results reflect the average value of signals in a group of cells, or only represent the dominant number of cells, resulting in the neglect of heterogeneity information between different cells. Single cell sequencing can detect the heterogeneity of single cells, identify rare cells, and reveal the differences between cells. [4]

Fig. 2 Schematic diagram of comparison between single cell sequencing (top) and traditional mixed cell sequencing (bottom)

Single cell sequencing technology发展

After Tang Fuhui and others completed the first mammalian single-cell RNA transcriptome sequencing in 2009, single-cell sequencing has experienced rapid development for more than ten years. At the same time, with the update and iteration of sequencing technology, single-cell analysis systems developed by various manufacturers based on different detection principles continue to introduce new ones, and single-cell sequencing has gradually realized the transformation from low-throughput to high-throughput detection. The official announcement of the”human cell Atlas (HCA)” in 2017 is an important milestone in the industrialization of high-throughput single-cell research.

Figure 3 major development process of single cell research [5]

Single cell sequencing technology流程

At first, single-cell sequencing used different methods to separate single cells and independently construct a library for sequencing. However, this method has low cell flux (only detecting dozens of cells and not enough to reflect the real situation) and high cost.

With the development of sequencing technology, a single cell recognition technology based on barcode has emerged, that is, it does not need to separate single cells, just add a separate tag sequence to each cell, and sequence it by building a database at one time. This method makes single cell sequencing enter a high-throughput era, and the cost of single cell separation and sequencing is greatly reduced.

与传统混合细胞测序不同的是,单细胞测序起始样本中核酸含量极低,需要对筛选出的细胞扩增后才能满足后期测序实验,目标是在尽量减少序列扩增偏差的前提下增加核酸总量利于后续分析。Single cell sequencing technology操作流程包括:样本细胞筛选、核酸提取及扩增、测序文库构建、测序和数据分析。

Fig. 4 Schematic diagram of comparison between single cell sequencing (top) and traditional hybrid cell sequencing (bottom) technical processes

reference

[1] Tammela,Tuomas; Sage,Julien (2020).  “Investigating Tumor Heterogeneity in Mouse Models”.  Annual Review of Cancer Biology.  4(1):99–119. doi:10.1146/annurev-cancerbio-030419-033413.

[2] Zemach A, McDaniel IE, Silva P, Zilberman D (May 2010).  “Genome-wide evolutionary analysis of eukaryotic DNA methylation”.  Science.  328 (5980):916-9.  Bibcode:2010Sci… 328.. 916Z.  doi:10.1126/science. 1186366. 

[3] Jialong Liang , Wanshi Cai , Zhongsheng Sun. Single-Cell Sequencing Technologies:Current and Future[J]. Journal of Genetics and Genomics 41 (2014) 513-528

[4] Eberwine J, Sul JY, Bartfai T, Kim J ,The promise of single-cell sequencing[J].  Nature Methods.  2014,11 (1):25–7.  doi:10.1038/nmeth. two thousand seven hundred and sixty-nine

[5] Gene Hui”2020 single cell research report”