Abstract<\/h2>\n\nGene heterogeneity leads to the differences in cellular behaviors in a wide range, such as tumor drug-resistant mutation, epithelial-mesenchymal transition, and migration, posing significant challenges to the development of biomedicine. Traditional gene analysis methods, such as polymerase chain reaction, employ a mass of cells as the gene source, resulting in that the gene properties from a specific single cell are hidden in massive gene information. Recent decades have seen the emerging single-cell gene analysis techniques with their unprecedented opportunities to study gene heterogeneity with high precision and high throughput. In this review, we summarized the state-of-the-art techniques for single-cell sequencing and on-chip gene analysis systems. The principles of each technique are introduced in detail, with the focus on the application scenarios in gene heterogeneity research. Looking forward, we also introduced the challenges in current technologies and point out the future direction for facilitating the technical improvement and clinical applications of single-cell gene analysis techniques.<\/p>\n<\/div>\n<\/section>\n<\/div>\n
<\/div>\n\n\n1 INTRODUCTION<\/h2>\n
Gene heterogeneity inherently exists among different single cells owing to that stochastic changes constantly occur in the process of genome replication, cell division, and gene transcription.1<\/a>,\u00a02<\/a><\/sup>\u00a0However, the ever-changing gene-phenotype brings unpredictable risks for the exploration of cell mechanisms and the precise treatment of diseases.3<\/a>–5<\/a><\/sup>\u00a0Fully understanding the subtle gene differences among single cells is crucial for decoding gene heterogeneity.6<\/a><\/sup>\u00a0In the past decades, conventional genetic analysis methods in cytobiology, such as polymerase chain reaction (PCR) and gel electrophoresis, primarily take bulk populations of cells as analytes, which neglect the specific gene phenotypes from single cells. For the purpose of gene heterogeneity research, there is an urgent desire to accurately interrogate these distinctive subpopulations at the single-cell level.7<\/a>,\u00a08<\/a><\/sup><\/p>\nIn recent years, advanced cytobiology and bioengineering technologies have offered alternative choices to realize gene analysis at the single-cell level. From the point of view of cytobiology, single-cell sequencing technologies have been widely applied to uncover molecular mechanisms, analyze gene multi-omics, and classify tumor subtypes.9<\/a>–12<\/a><\/sup>\u00a0For instance, nanopore sequencing technology has the capability of ultra-long reading length without the limitation of gene labels, which attracts the attention of researchers in single-cell gene sequencing.13<\/a>,\u00a014<\/a><\/sup>\u00a0In the field of bioengineering, the micro-electromechanical systems make the design of Lab-on-Chip systems available for single-cell analysis with marked advantages, including accelerated efficiency, automation, and ultrahigh throughput.15<\/a>–17<\/a><\/sup>\u00a0Especially, the on-chip single living cell gene analysis enables in-situ insight into the dynamic gene properties and real-time cell behaviors.18<\/a>–20<\/a><\/sup>\u00a0To address the limitations of gene content from single cells,21<\/a><\/sup>\u00a0researchers in cytobiology are gradually forming alliances with bioengineering scientists to develop advanced single-cell gene analysis technologies for gene heterogeneity research.<\/p>\nIn this review, we systematically introduced the recent advances in both off-chip single-cell sequencing techniques and on-chip single-cell gene analysis systems (Figure\u00a01<\/a>). We summarized the recent progress of single-cell sequencing from three types of cellular omics, that is, genome, transcriptome, and epigenome. Furthermore, the novel on-chip bioanalytical systems for the high-throughput cell manipulation and the interrogation of single-cell genes are introduced in detail according to their structural features. We also discussed the current challenges faced in single-cell gene analysis, as well as the future opportunities for clinical application. This review comprehensively exhibits the advanced single-cell gene analysis techniques in cytology and bioengineering, expecting to promote the study of gene heterogeneity and provide references for precision medicine.<\/p>\n\n![\"Details](\"https:\/\/onlinelibrary.wiley.com\/cms\/asset\/1b2d9f6b-f00d-481d-8363-d72e09759dfd\/viw2191-fig-0001-m.png\")
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Gene heterogeneity leads to the differences in cellular behaviors in a wide range, such as tumor drug-resistant mutation, epithelial-mesenchymal transition, and migration, posing significant challenges to the development of biomedicine. Traditional gene analysis methods, such as polymerase chain reaction, employ a mass of cells as the gene source, resulting in that the gene properties from a specific single cell are hidden in massive gene information. Recent decades have seen the emerging single-cell gene analysis techniques with their unprecedented opportunities to study gene heterogeneity with high precision and high throughput. In this review, we summarized the state-of-the-art techniques for single-cell sequencing and on-chip gene analysis systems. The principles of each technique are introduced in detail, with the focus on the application scenarios in gene heterogeneity research. Looking forward, we also introduced the challenges in current technologies and point out the future direction for facilitating the technical improvement and clinical applications of single-cell gene analysis techniques.<\/p>\n<\/div>\n<\/section>\n<\/div>\n
1 INTRODUCTION<\/h2>\n
Gene heterogeneity inherently exists among different single cells owing to that stochastic changes constantly occur in the process of genome replication, cell division, and gene transcription.1<\/a>,\u00a02<\/a><\/sup>\u00a0However, the ever-changing gene-phenotype brings unpredictable risks for the exploration of cell mechanisms and the precise treatment of diseases.3<\/a>–5<\/a><\/sup>\u00a0Fully understanding the subtle gene differences among single cells is crucial for decoding gene heterogeneity.6<\/a><\/sup>\u00a0In the past decades, conventional genetic analysis methods in cytobiology, such as polymerase chain reaction (PCR) and gel electrophoresis, primarily take bulk populations of cells as analytes, which neglect the specific gene phenotypes from single cells. For the purpose of gene heterogeneity research, there is an urgent desire to accurately interrogate these distinctive subpopulations at the single-cell level.7<\/a>,\u00a08<\/a><\/sup><\/p>\n In recent years, advanced cytobiology and bioengineering technologies have offered alternative choices to realize gene analysis at the single-cell level. From the point of view of cytobiology, single-cell sequencing technologies have been widely applied to uncover molecular mechanisms, analyze gene multi-omics, and classify tumor subtypes.9<\/a>–12<\/a><\/sup>\u00a0For instance, nanopore sequencing technology has the capability of ultra-long reading length without the limitation of gene labels, which attracts the attention of researchers in single-cell gene sequencing.13<\/a>,\u00a014<\/a><\/sup>\u00a0In the field of bioengineering, the micro-electromechanical systems make the design of Lab-on-Chip systems available for single-cell analysis with marked advantages, including accelerated efficiency, automation, and ultrahigh throughput.15<\/a>–17<\/a><\/sup>\u00a0Especially, the on-chip single living cell gene analysis enables in-situ insight into the dynamic gene properties and real-time cell behaviors.18<\/a>–20<\/a><\/sup>\u00a0To address the limitations of gene content from single cells,21<\/a><\/sup>\u00a0researchers in cytobiology are gradually forming alliances with bioengineering scientists to develop advanced single-cell gene analysis technologies for gene heterogeneity research.<\/p>\n In this review, we systematically introduced the recent advances in both off-chip single-cell sequencing techniques and on-chip single-cell gene analysis systems (Figure\u00a01<\/a>). We summarized the recent progress of single-cell sequencing from three types of cellular omics, that is, genome, transcriptome, and epigenome. Furthermore, the novel on-chip bioanalytical systems for the high-throughput cell manipulation and the interrogation of single-cell genes are introduced in detail according to their structural features. We also discussed the current challenges faced in single-cell gene analysis, as well as the future opportunities for clinical application. This review comprehensively exhibits the advanced single-cell gene analysis techniques in cytology and bioengineering, expecting to promote the study of gene heterogeneity and provide references for precision medicine.<\/p>\n![\"Details](\"\/cms\/asset\/83835423-02c7-48c5-84eb-7b77409e47ba\/viw2191-fig-0001-m.jpg\")
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