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With the rapid development of biotechnologies, single-cell sequencing has become an important tool for understanding the molecular mechanisms of diseases, defining cellular heterogeneities and characteristics, and identifying intercellular communications and single-cell-based biomarkers. Providing a clear overview of the clinical applications, the book presents state-of-the-art information on immune cell function, cancer progression, infection, and inflammation gained from single-cell DNA or RNA sequencing. Furthermore, it explores the role of target gene methylation in the pathogenesis of diseases, with a focus on respiratory cancer, infection and chronic diseases. As such it is a valuable resource for clinical researchers and physicians, allowing them to refresh their knowledge and improve early diagnosis and therapy for patients.
Book Synopsis Single-cell Sequencing and Methylation by : Buwei Yu
Download or read book Single-cell Sequencing and Methylation written by Buwei Yu and published by Springer Nature. This book was released on 2020-09-19 with total page 247 pages. Available in PDF, EPUB and Kindle. Book excerpt: With the rapid development of biotechnologies, single-cell sequencing has become an important tool for understanding the molecular mechanisms of diseases, defining cellular heterogeneities and characteristics, and identifying intercellular communications and single-cell-based biomarkers. Providing a clear overview of the clinical applications, the book presents state-of-the-art information on immune cell function, cancer progression, infection, and inflammation gained from single-cell DNA or RNA sequencing. Furthermore, it explores the role of target gene methylation in the pathogenesis of diseases, with a focus on respiratory cancer, infection and chronic diseases. As such it is a valuable resource for clinical researchers and physicians, allowing them to refresh their knowledge and improve early diagnosis and therapy for patients.
DNA methylation at cytosines has long been associated with early development, maturation, and aging of human tissues. Traditionally, DNA methylation is associated with gene silencing. However, recent single cell multi-omic DNA methylation and RNA sequencing methods have shown that the role of DNA methylation on the expression of nearby genes could silence or activate them depending on the gene and cell type. The recent developments these assays have detected cell type specific DNA methylation and RNA coupling in stem cell rich and human brain tissues. This specificity underscores the need for future growth in DNA methylation and RNA co-sequencing technologies and analysis tools. Presently, about 100,000 single cell profiles are required to adequately map tissues. DNA methylation and RNA co-sequencing methods require the physical isolation of single cells in individual wells. There is no method that can assay 100,000 cells without utilizing extensive liquid handling systems. We address this challenge by developing a novel ultra-high throughput DNA methylation and RNA co-sequencing platform, sci-Gel, that utilizes three levels of combinatorial indexing to increase the throughput of existing technologies to 50,000-100,000 cells per experiment with just three 96 well plates. In this dissertation, we first push the boundaries of present combinatorial indexing techniques where the DNA and RNA of single cells are simultaneously extracted and immobilized within polyacrylamide gel beads that are used for indexing. This resulted in the development of a 2-level combinatorial indexing platform that could be used to co-sequence DNA copy-number variations, relevant in cancers, and RNA at the scale of thousands of cells. We then describe the adaptations made from existing bisulfite conversion chemistries to our gel beads to incorporate the DNA methylation feature. We then describe the development of a 3-level combinatorial indexing platform to increase the cell throughput of our technology to 50,000-100,000 cells per experiment. Finally, we discuss future efforts to utilize sci-Gel to create the first single cell DNA methylation and RNA co-sequencing map of peripheral blood mononuclear cells.
Book Synopsis Ultra-High Throughput Single Cell Co-Sequencing of DNA Methylation and RNA Using 3-Level Combinatorial Indexing by : Huy Lam
Download or read book Ultra-High Throughput Single Cell Co-Sequencing of DNA Methylation and RNA Using 3-Level Combinatorial Indexing written by Huy Lam and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA methylation at cytosines has long been associated with early development, maturation, and aging of human tissues. Traditionally, DNA methylation is associated with gene silencing. However, recent single cell multi-omic DNA methylation and RNA sequencing methods have shown that the role of DNA methylation on the expression of nearby genes could silence or activate them depending on the gene and cell type. The recent developments these assays have detected cell type specific DNA methylation and RNA coupling in stem cell rich and human brain tissues. This specificity underscores the need for future growth in DNA methylation and RNA co-sequencing technologies and analysis tools. Presently, about 100,000 single cell profiles are required to adequately map tissues. DNA methylation and RNA co-sequencing methods require the physical isolation of single cells in individual wells. There is no method that can assay 100,000 cells without utilizing extensive liquid handling systems. We address this challenge by developing a novel ultra-high throughput DNA methylation and RNA co-sequencing platform, sci-Gel, that utilizes three levels of combinatorial indexing to increase the throughput of existing technologies to 50,000-100,000 cells per experiment with just three 96 well plates. In this dissertation, we first push the boundaries of present combinatorial indexing techniques where the DNA and RNA of single cells are simultaneously extracted and immobilized within polyacrylamide gel beads that are used for indexing. This resulted in the development of a 2-level combinatorial indexing platform that could be used to co-sequence DNA copy-number variations, relevant in cancers, and RNA at the scale of thousands of cells. We then describe the adaptations made from existing bisulfite conversion chemistries to our gel beads to incorporate the DNA methylation feature. We then describe the development of a 3-level combinatorial indexing platform to increase the cell throughput of our technology to 50,000-100,000 cells per experiment. Finally, we discuss future efforts to utilize sci-Gel to create the first single cell DNA methylation and RNA co-sequencing map of peripheral blood mononuclear cells.
This volume provides a comprehensive overview for investigating biology at the level of individual cells. Chapters are organized into eight parts detailing a single-cell lab, single cell DNA-seq, RNA-seq, single cell proteomic and epigenetic, single cell multi-omics, single cell screening, and single cell live imaging. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Single Cell Methods: Sequencing and Proteomics aims to make each experiment easily reproducible in every lab.
Book Synopsis Single Cell Methods by : Valentina Proserpio
Download or read book Single Cell Methods written by Valentina Proserpio and published by . This book was released on 2019 with total page 452 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume provides a comprehensive overview for investigating biology at the level of individual cells. Chapters are organized into eight parts detailing a single-cell lab, single cell DNA-seq, RNA-seq, single cell proteomic and epigenetic, single cell multi-omics, single cell screening, and single cell live imaging. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Single Cell Methods: Sequencing and Proteomics aims to make each experiment easily reproducible in every lab.
Single-cell omics is a progressing frontier that stems from the sequencing of the human genome and the development of omics technologies, particularly genomics, transcriptomics, epigenomics and proteomics, but the sensitivity is now improved to single-cell level. The new generation of methodologies, especially the next generation sequencing (NGS) technology, plays a leading role in genomics related fields; however, the conventional techniques of omics require number of cells to be large, usually on the order of millions of cells, which is hardly accessible in some cases. More importantly, harnessing the power of omics technologies and applying those at the single-cell level are crucial since every cell is specific and unique, and almost every cell population in every systems, derived in either vivo or in vitro, is heterogeneous. Deciphering the heterogeneity of the cell population hence becomes critical for recognizing the mechanism and significance of the system. However, without an extensive examination of individual cells, a massive analysis of cell population would only give an average output of the cells, but neglect the differences among cells. Single-cell omics seeks to study a number of individual cells in parallel for their different dimensions of molecular profile on genome-wide scale, providing unprecedented resolution for the interpretation of both the structure and function of an organ, tissue or other system, as well as the interaction (and communication) and dynamics of single cells or subpopulations of cells and their lineages. Importantly single-cell omics enables the identification of a minor subpopulation of cells that may play a critical role in biological process over a dominant subpolulation such as a cancer and a developing organ. It provides an ultra-sensitive tool for us to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. Besides, it also empowers the clinical investigation of patients when facing a very low quantity of cell available for analysis, such as noninvasive cancer screening with circulating tumor cells (CTC), noninvasive prenatal diagnostics (NIPD) and preimplantation genetic test (PGT) for in vitro fertilization. Single-cell omics greatly promotes the understanding of life at a more fundamental level, bring vast applications in medicine. Accordingly, single-cell omics is also called as single-cell analysis or single-cell biology. Within only a couple of years, single-cell omics, especially transcriptomic sequencing (scRNA-seq), whole genome and exome sequencing (scWGS, scWES), has become robust and broadly accessible. Besides the existing technologies, recently, multiplexing barcode design and combinatorial indexing technology, in combination with microfluidic platform exampled by Drop-seq, or even being independent of microfluidic platform but using a regular PCR-plate, enable us a greater capacity of single cell analysis, switching from one single cell to thousands of single cells in a single test. The unique molecular identifiers (UMIs) allow the amplification bias among the original molecules to be corrected faithfully, resulting in a reliable quantitative measurement of omics in single cells. Of late, a variety of single-cell epigenomics analyses are becoming sophisticated, particularly single cell chromatin accessibility (scATAC-seq) and CpG methylation profiling (scBS-seq, scRRBS-seq). High resolution single molecular Fluorescence in situ hybridization (smFISH) and its revolutionary versions (ex. seqFISH, MERFISH, and so on), in addition to the spatial transcriptome sequencing, make the native relationship of the individual cells of a tissue to be in 3D or 4D format visually and quantitatively clarified. On the other hand, CRISPR/cas9 editing-based In vivo lineage tracing methods enable dynamic profile of a whole developmental process to be accurately displayed. Multi-omics analysis facilitates the study of multi-dimensional regulation and relationship of different elements of the central dogma in a single cell, as well as permitting a clear dissection of the complicated omics heterogeneity of a system. Last but not the least, the technology, biological noise, sequence dropout, and batch effect bring a huge challenge to the bioinformatics of single cell omics. While significant progress in the data analysis has been made since then, revolutionary theory and algorithm logics for single cell omics are expected. Indeed, single-cell analysis exert considerable impacts on the fields of biological studies, particularly cancers, neuron and neural system, stem cells, embryo development and immune system; other than that, it also tremendously motivates pharmaceutic RD, clinical diagnosis and monitoring, as well as precision medicine. This book hereby summarizes the recent developments and general considerations of single-cell analysis, with a detailed presentation on selected technologies and applications. Starting with the experimental design on single-cell omics, the book then emphasizes the consideration on heterogeneity of cancer and other systems. It also gives an introduction of the basic methods and key facts for bioinformatics analysis. Secondary, this book provides a summary of two types of popular technologies, the fundamental tools on single-cell isolation, and the developments of single cell multi-omics, followed by descriptions of FISH technologies, though other popular technologies are not covered here due to the fact that they are intensively described here and there recently. Finally, the book illustrates an elastomer-based integrated fluidic circuit that allows a connection between single cell functional studies combining stimulation, response, imaging and measurement, and corresponding single cell sequencing. This is a model system for single cell functional genomics. In addition, it reports a pipeline for single-cell proteomics with an analysis of the early development of Xenopus embryo, a single-cell qRT-PCR application that defined the subpopulations related to cell cycling, and a new method for synergistic assembly of single cell genome with sequencing of amplification product by phi29 DNA polymerase. Due to the tremendous progresses of single-cell omics in recent years, the topics covered here are incomplete, but each individual topic is excellently addressed, significantly interesting and beneficial to scientists working in or affiliated with this field.
Book Synopsis Introduction to Single Cell Omics by : Xinghua Pan
Download or read book Introduction to Single Cell Omics written by Xinghua Pan and published by Frontiers Media SA. This book was released on 2019-09-19 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell omics is a progressing frontier that stems from the sequencing of the human genome and the development of omics technologies, particularly genomics, transcriptomics, epigenomics and proteomics, but the sensitivity is now improved to single-cell level. The new generation of methodologies, especially the next generation sequencing (NGS) technology, plays a leading role in genomics related fields; however, the conventional techniques of omics require number of cells to be large, usually on the order of millions of cells, which is hardly accessible in some cases. More importantly, harnessing the power of omics technologies and applying those at the single-cell level are crucial since every cell is specific and unique, and almost every cell population in every systems, derived in either vivo or in vitro, is heterogeneous. Deciphering the heterogeneity of the cell population hence becomes critical for recognizing the mechanism and significance of the system. However, without an extensive examination of individual cells, a massive analysis of cell population would only give an average output of the cells, but neglect the differences among cells. Single-cell omics seeks to study a number of individual cells in parallel for their different dimensions of molecular profile on genome-wide scale, providing unprecedented resolution for the interpretation of both the structure and function of an organ, tissue or other system, as well as the interaction (and communication) and dynamics of single cells or subpopulations of cells and their lineages. Importantly single-cell omics enables the identification of a minor subpopulation of cells that may play a critical role in biological process over a dominant subpolulation such as a cancer and a developing organ. It provides an ultra-sensitive tool for us to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. Besides, it also empowers the clinical investigation of patients when facing a very low quantity of cell available for analysis, such as noninvasive cancer screening with circulating tumor cells (CTC), noninvasive prenatal diagnostics (NIPD) and preimplantation genetic test (PGT) for in vitro fertilization. Single-cell omics greatly promotes the understanding of life at a more fundamental level, bring vast applications in medicine. Accordingly, single-cell omics is also called as single-cell analysis or single-cell biology. Within only a couple of years, single-cell omics, especially transcriptomic sequencing (scRNA-seq), whole genome and exome sequencing (scWGS, scWES), has become robust and broadly accessible. Besides the existing technologies, recently, multiplexing barcode design and combinatorial indexing technology, in combination with microfluidic platform exampled by Drop-seq, or even being independent of microfluidic platform but using a regular PCR-plate, enable us a greater capacity of single cell analysis, switching from one single cell to thousands of single cells in a single test. The unique molecular identifiers (UMIs) allow the amplification bias among the original molecules to be corrected faithfully, resulting in a reliable quantitative measurement of omics in single cells. Of late, a variety of single-cell epigenomics analyses are becoming sophisticated, particularly single cell chromatin accessibility (scATAC-seq) and CpG methylation profiling (scBS-seq, scRRBS-seq). High resolution single molecular Fluorescence in situ hybridization (smFISH) and its revolutionary versions (ex. seqFISH, MERFISH, and so on), in addition to the spatial transcriptome sequencing, make the native relationship of the individual cells of a tissue to be in 3D or 4D format visually and quantitatively clarified. On the other hand, CRISPR/cas9 editing-based In vivo lineage tracing methods enable dynamic profile of a whole developmental process to be accurately displayed. Multi-omics analysis facilitates the study of multi-dimensional regulation and relationship of different elements of the central dogma in a single cell, as well as permitting a clear dissection of the complicated omics heterogeneity of a system. Last but not the least, the technology, biological noise, sequence dropout, and batch effect bring a huge challenge to the bioinformatics of single cell omics. While significant progress in the data analysis has been made since then, revolutionary theory and algorithm logics for single cell omics are expected. Indeed, single-cell analysis exert considerable impacts on the fields of biological studies, particularly cancers, neuron and neural system, stem cells, embryo development and immune system; other than that, it also tremendously motivates pharmaceutic RD, clinical diagnosis and monitoring, as well as precision medicine. This book hereby summarizes the recent developments and general considerations of single-cell analysis, with a detailed presentation on selected technologies and applications. Starting with the experimental design on single-cell omics, the book then emphasizes the consideration on heterogeneity of cancer and other systems. It also gives an introduction of the basic methods and key facts for bioinformatics analysis. Secondary, this book provides a summary of two types of popular technologies, the fundamental tools on single-cell isolation, and the developments of single cell multi-omics, followed by descriptions of FISH technologies, though other popular technologies are not covered here due to the fact that they are intensively described here and there recently. Finally, the book illustrates an elastomer-based integrated fluidic circuit that allows a connection between single cell functional studies combining stimulation, response, imaging and measurement, and corresponding single cell sequencing. This is a model system for single cell functional genomics. In addition, it reports a pipeline for single-cell proteomics with an analysis of the early development of Xenopus embryo, a single-cell qRT-PCR application that defined the subpopulations related to cell cycling, and a new method for synergistic assembly of single cell genome with sequencing of amplification product by phi29 DNA polymerase. Due to the tremendous progresses of single-cell omics in recent years, the topics covered here are incomplete, but each individual topic is excellently addressed, significantly interesting and beneficial to scientists working in or affiliated with this field.
This book presents an overview of the recent technologies in single molecule and single cell sequencing. These sequencing technologies are revolutionizing the way of the genomic studies and the understanding of complex biological systems. The PacBio sequencer has enabled extremely long-read sequencing and the MinION sequencer has made the sequencing possible in developing countries. New developments and technologies are constantly emerging, which will further expand sequencing applications. In parallel, single cell sequencing technologies are rapidly becoming a popular platform. This volume presents not only an updated overview of these technologies, but also of the related developments in bioinformatics. Without powerful bioinformatics software, where rapid progress is taking place, these new technologies will not realize their full potential. All the contributors to this volume have been involved in the development of these technologies and software and have also made significant progress on their applications. This book is intended to be of interest to a wide audience ranging from genome researchers to basic molecular biologists and clinicians.
Book Synopsis Single Molecule and Single Cell Sequencing by : Yutaka Suzuki
Download or read book Single Molecule and Single Cell Sequencing written by Yutaka Suzuki and published by Springer. This book was released on 2019-04-09 with total page 150 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents an overview of the recent technologies in single molecule and single cell sequencing. These sequencing technologies are revolutionizing the way of the genomic studies and the understanding of complex biological systems. The PacBio sequencer has enabled extremely long-read sequencing and the MinION sequencer has made the sequencing possible in developing countries. New developments and technologies are constantly emerging, which will further expand sequencing applications. In parallel, single cell sequencing technologies are rapidly becoming a popular platform. This volume presents not only an updated overview of these technologies, but also of the related developments in bioinformatics. Without powerful bioinformatics software, where rapid progress is taking place, these new technologies will not realize their full potential. All the contributors to this volume have been involved in the development of these technologies and software and have also made significant progress on their applications. This book is intended to be of interest to a wide audience ranging from genome researchers to basic molecular biologists and clinicians.
Next generation sequencing (NGS) has surpassed the traditional Sanger sequencing method to become the main choice for large-scale, genome-wide sequencing studies with ultra-high-throughput production and a huge reduction in costs. The NGS technologies have had enormous impact on the studies of structural and functional genomics in all the life sciences. In this book, Next Generation Sequencing Advances, Applications and Challenges, the sixteen chapters written by experts cover various aspects of NGS including genomics, transcriptomics and methylomics, the sequencing platforms, and the bioinformatics challenges in processing and analysing huge amounts of sequencing data. Following an overview of the evolution of NGS in the brave new world of omics, the book examines the advances and challenges of NGS applications in basic and applied research on microorganisms, agricultural plants and humans. This book is of value to all who are interested in DNA sequencing and bioinformatics across all fields of the life sciences.
Book Synopsis Next Generation Sequencing by : Jerzy Kulski
Download or read book Next Generation Sequencing written by Jerzy Kulski and published by BoD – Books on Demand. This book was released on 2016-01-14 with total page 466 pages. Available in PDF, EPUB and Kindle. Book excerpt: Next generation sequencing (NGS) has surpassed the traditional Sanger sequencing method to become the main choice for large-scale, genome-wide sequencing studies with ultra-high-throughput production and a huge reduction in costs. The NGS technologies have had enormous impact on the studies of structural and functional genomics in all the life sciences. In this book, Next Generation Sequencing Advances, Applications and Challenges, the sixteen chapters written by experts cover various aspects of NGS including genomics, transcriptomics and methylomics, the sequencing platforms, and the bioinformatics challenges in processing and analysing huge amounts of sequencing data. Following an overview of the evolution of NGS in the brave new world of omics, the book examines the advances and challenges of NGS applications in basic and applied research on microorganisms, agricultural plants and humans. This book is of value to all who are interested in DNA sequencing and bioinformatics across all fields of the life sciences.
High throughput sequencing (HTS) technologies have conquered the genomics and epigenomics worlds. The applications of HTS methods are wide, and can be used to sequence everything from whole or partial genomes, transcriptomes, non-coding RNAs, ribosome profiling, to single-cell sequencing. Having such diversity of alternatives, there is a demand for information by research scientists without experience in HTS that need to choose the most suitable methodology or combination of platforms and to define their experimental designs to achieve their specific objectives. Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing aims to collect in a single volume all aspects that should be taken into account when HTS technologies are being incorporated into a research project and the reasons behind them. Moreover, examples of several successful strategies will be analyzed to make the point of the crucial features. This book will be of use to all scientist that are unfamiliar with HTS and want to incorporate such technologies to their research.
Book Synopsis Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing by : Ana M. Aransay
Download or read book Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing written by Ana M. Aransay and published by Springer. This book was released on 2016-06-02 with total page 404 pages. Available in PDF, EPUB and Kindle. Book excerpt: High throughput sequencing (HTS) technologies have conquered the genomics and epigenomics worlds. The applications of HTS methods are wide, and can be used to sequence everything from whole or partial genomes, transcriptomes, non-coding RNAs, ribosome profiling, to single-cell sequencing. Having such diversity of alternatives, there is a demand for information by research scientists without experience in HTS that need to choose the most suitable methodology or combination of platforms and to define their experimental designs to achieve their specific objectives. Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing aims to collect in a single volume all aspects that should be taken into account when HTS technologies are being incorporated into a research project and the reasons behind them. Moreover, examples of several successful strategies will be analyzed to make the point of the crucial features. This book will be of use to all scientist that are unfamiliar with HTS and want to incorporate such technologies to their research.
This book presents the latest advances in precision medicine in some of the most common cancer types, including hematological, lung and breast malignancies. It also discusses emerging technologies that are making a significant impact on precision medicine in cancer therapy. In addition to describing specific approaches that have already entered clinical practice, the book explores new concepts and tools that are being developed. Precision medicine aims to deliver personalized healthcare tailored to a patient’s genetics, lifestyle and environment, and cancer therapy is one of the areas in which it has flourished in recent years. Documenting the latest advances, this book is of interest to physicians and clinical fellows in the front line of the war on cancer, as well as to basic scientists working in the fields of cancer biology, drug development, biomarker discovery, and biomedical engineering. The contributing authors include translational physicians with first-hand experience in precision patient care.
Book Synopsis Precision Medicine in Cancer Therapy by : Daniel D. Von Hoff
Download or read book Precision Medicine in Cancer Therapy written by Daniel D. Von Hoff and published by Springer. This book was released on 2019-06-17 with total page 283 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents the latest advances in precision medicine in some of the most common cancer types, including hematological, lung and breast malignancies. It also discusses emerging technologies that are making a significant impact on precision medicine in cancer therapy. In addition to describing specific approaches that have already entered clinical practice, the book explores new concepts and tools that are being developed. Precision medicine aims to deliver personalized healthcare tailored to a patient’s genetics, lifestyle and environment, and cancer therapy is one of the areas in which it has flourished in recent years. Documenting the latest advances, this book is of interest to physicians and clinical fellows in the front line of the war on cancer, as well as to basic scientists working in the fields of cancer biology, drug development, biomarker discovery, and biomedical engineering. The contributing authors include translational physicians with first-hand experience in precision patient care.
This book provides an overview of single-cell isolation, separation, injection, lysis and dynamics analysis as well as a study of their heterogeneity using different miniaturized devices. As an important part of single-cell analysis, different techniques including electroporation, microinjection, optical trapping, optoporation, rapid electrokinetic patterning and optoelectronic tweezers are described in detail. It presents different fluidic systems (e.g. continuous micro/nano-fluidic devices, microfluidic cytometry) and their integration with sensor technology, optical and hydrodynamic stretchers etc., and demonstrates the applications of single-cell analysis in systems biology, proteomics, genomics, epigenomics, cancer transcriptomics, metabolomics, biomedicine and drug delivery systems. It also discusses the future challenges for single-cell analysis, including the advantages and limitations. This book is enjoyable reading material while at the same time providing essential information to scientists in academia and professionals in industry working on different aspects of single-cell analysis. Dr. Fan-Gang Tseng is a Distinguished Professor of Engineering and System Science at the National Tsing Hua University, Taiwan. Dr. Tuhin Subhra Santra is a Research Associate at the California Nano Systems Institute, University of California at Los Angeles, USA.
Book Synopsis Essentials of Single-Cell Analysis by : Fan-Gang Tseng
Download or read book Essentials of Single-Cell Analysis written by Fan-Gang Tseng and published by Springer. This book was released on 2016-01-21 with total page 415 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides an overview of single-cell isolation, separation, injection, lysis and dynamics analysis as well as a study of their heterogeneity using different miniaturized devices. As an important part of single-cell analysis, different techniques including electroporation, microinjection, optical trapping, optoporation, rapid electrokinetic patterning and optoelectronic tweezers are described in detail. It presents different fluidic systems (e.g. continuous micro/nano-fluidic devices, microfluidic cytometry) and their integration with sensor technology, optical and hydrodynamic stretchers etc., and demonstrates the applications of single-cell analysis in systems biology, proteomics, genomics, epigenomics, cancer transcriptomics, metabolomics, biomedicine and drug delivery systems. It also discusses the future challenges for single-cell analysis, including the advantages and limitations. This book is enjoyable reading material while at the same time providing essential information to scientists in academia and professionals in industry working on different aspects of single-cell analysis. Dr. Fan-Gang Tseng is a Distinguished Professor of Engineering and System Science at the National Tsing Hua University, Taiwan. Dr. Tuhin Subhra Santra is a Research Associate at the California Nano Systems Institute, University of California at Los Angeles, USA.
Single-cell genomics is the study of molecular modalities, or "-omes" from individual cells. Many protocols have been developed to profile genome, epigenome, transcriptome, and proteome from single cells. Among all these protocols, single-cell transcriptome profiling using single-cell RNA sequencing is the most popular and mature one. This technique has been demonstrated to be very powerful in dissecting cell types within a heterogeneous tissue, as well as revealing cell type specific responses to stimuli. It has also been used to reconstruct cell trajectory during complex biological progress, such as cell differentiation. More importantly, it can be used to reveal gene co-expression networks among cell types, and ultimately the molecular mechanism of gene regulation. In the first two projects of my thesis, I described how we use single-cell RNA sequencing to understand the molecular mechanism controlling trophoblasts proliferation and differentiation during human peri-implantation embryo development, as well as the mechanism of retinal progenitor cells commitment during early human retinogenesis. In the first project, we profiled human embryonic stem cell derived retinal organoids using single-cell RNA sequencing, to understand the molecular mechanism of early retinogenesis. The development of the mammalian retina is a complicated process involving generating distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA-sequencing (scRNA-seq) on cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinct subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found genes related to the Notch and Wnt signaling pathway, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified CCND1, a G1-phase cell cycle regulator, was co-expressed with ASCL1 in a cell-cycle independent manner. Temporally-controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in the early retinogenesis of humans. In the second project, we profiled transcriptome from individual trophoblast cells collected from human peri-implantation embryos, to reveal how these cells proliferate and differentiate to establish placenta. Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after conceptus implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development in human remains elusive. In this study, we modeled human conceptus peri-implantation development from blastocyst to early post-implantation stages by using an in vitro coculture system and profiled the transcriptome of 476 individual trophoblast cells from these conceptuses. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast into syncytiotrophoblast. The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development. In parallel with the development of single-cell RNA sequencing, many efforts have been put in profiling other molecular modalities from single cells, such as genome, epigenome, and proteome. By elaborately combining these protocols, we can profile more than one types of "omes" from individual cells simultaneously. These techniques, commonly termed as "single-cell multimodal profiling", can generate data that has certain advantages compared to single-cell "mono-omics" approaches. Specifically, since each molecular modality provides orthogonal information about cell identities and status, the joint clustering of single-cell multi-omics data can better resolve cell types within a heterogeneous cell population. Also, because more than one molecular modalities were profiled simultaneously from every single cell, we could have better inferences about the relationship between these omics. In the third project, we demonstrated how to use scMT-seq (simultaneous profiling of transcriptome and DNA methylome from a single cell), to investigate the gene regulatory role of DNA methylation in sensory neurons during peripheral nerve injury response and regeneration. DNA methylation is implicated in neuronal injury response and regeneration, but its role in regulating stable transcription changes in different types of dorsal root ganglion (DRG) neurons is unclear. In this study, we simultaneously profiled both the DNA methylome and mRNA transcriptome from single DRG neurons at different ages under either control or peripheral nerve injury condition. We found that age-related expression changes in Notch signaling genes and methylation changes at Notch receptor binding sites are associated with the age-dependent decline in peripheral nerve regeneration potential. Moreover, selective hypomethylation of AP-1 complex binding sites on regeneration-associated gene (RAG) promoters coincides with RAG transcriptional upregulation after injury. Consistent with the findings that different subtypes of DRG neurons exhibit distinct methylome changes upon injury responses, in a hybrid CAST/Ei; C57BL/6 genetic background, we further observed allele-specific gene regulation and methylation changes for many RAGs after injury. We suggest that the genetic background determines distinct allele-specific DNA methylomes, which contribute to age-dependent regulation and neuronal subtype-specific injury-responses in different mouse strains.
Book Synopsis The Applications of Single-cell Genomic Analysis in Development and Disease by : QIN AN
Download or read book The Applications of Single-cell Genomic Analysis in Development and Disease written by QIN AN and published by . This book was released on 2020 with total page 221 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell genomics is the study of molecular modalities, or "-omes" from individual cells. Many protocols have been developed to profile genome, epigenome, transcriptome, and proteome from single cells. Among all these protocols, single-cell transcriptome profiling using single-cell RNA sequencing is the most popular and mature one. This technique has been demonstrated to be very powerful in dissecting cell types within a heterogeneous tissue, as well as revealing cell type specific responses to stimuli. It has also been used to reconstruct cell trajectory during complex biological progress, such as cell differentiation. More importantly, it can be used to reveal gene co-expression networks among cell types, and ultimately the molecular mechanism of gene regulation. In the first two projects of my thesis, I described how we use single-cell RNA sequencing to understand the molecular mechanism controlling trophoblasts proliferation and differentiation during human peri-implantation embryo development, as well as the mechanism of retinal progenitor cells commitment during early human retinogenesis. In the first project, we profiled human embryonic stem cell derived retinal organoids using single-cell RNA sequencing, to understand the molecular mechanism of early retinogenesis. The development of the mammalian retina is a complicated process involving generating distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA-sequencing (scRNA-seq) on cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinct subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found genes related to the Notch and Wnt signaling pathway, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified CCND1, a G1-phase cell cycle regulator, was co-expressed with ASCL1 in a cell-cycle independent manner. Temporally-controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in the early retinogenesis of humans. In the second project, we profiled transcriptome from individual trophoblast cells collected from human peri-implantation embryos, to reveal how these cells proliferate and differentiate to establish placenta. Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after conceptus implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development in human remains elusive. In this study, we modeled human conceptus peri-implantation development from blastocyst to early post-implantation stages by using an in vitro coculture system and profiled the transcriptome of 476 individual trophoblast cells from these conceptuses. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast into syncytiotrophoblast. The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development. In parallel with the development of single-cell RNA sequencing, many efforts have been put in profiling other molecular modalities from single cells, such as genome, epigenome, and proteome. By elaborately combining these protocols, we can profile more than one types of "omes" from individual cells simultaneously. These techniques, commonly termed as "single-cell multimodal profiling", can generate data that has certain advantages compared to single-cell "mono-omics" approaches. Specifically, since each molecular modality provides orthogonal information about cell identities and status, the joint clustering of single-cell multi-omics data can better resolve cell types within a heterogeneous cell population. Also, because more than one molecular modalities were profiled simultaneously from every single cell, we could have better inferences about the relationship between these omics. In the third project, we demonstrated how to use scMT-seq (simultaneous profiling of transcriptome and DNA methylome from a single cell), to investigate the gene regulatory role of DNA methylation in sensory neurons during peripheral nerve injury response and regeneration. DNA methylation is implicated in neuronal injury response and regeneration, but its role in regulating stable transcription changes in different types of dorsal root ganglion (DRG) neurons is unclear. In this study, we simultaneously profiled both the DNA methylome and mRNA transcriptome from single DRG neurons at different ages under either control or peripheral nerve injury condition. We found that age-related expression changes in Notch signaling genes and methylation changes at Notch receptor binding sites are associated with the age-dependent decline in peripheral nerve regeneration potential. Moreover, selective hypomethylation of AP-1 complex binding sites on regeneration-associated gene (RAG) promoters coincides with RAG transcriptional upregulation after injury. Consistent with the findings that different subtypes of DRG neurons exhibit distinct methylome changes upon injury responses, in a hybrid CAST/Ei; C57BL/6 genetic background, we further observed allele-specific gene regulation and methylation changes for many RAGs after injury. We suggest that the genetic background determines distinct allele-specific DNA methylomes, which contribute to age-dependent regulation and neuronal subtype-specific injury-responses in different mouse strains.
