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Single-Cell Sequencing-Based Technologies Will Revolutionize Whole-Organism Science

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Single-Cell Sequencing-Based Technologies Will Revolutionize Whole-Organism Science REVIEWS APPLICATIONS OF NEXT-GENERATION SEQUENCING Single-cell sequencing-based technologies will revolutionize whole-organism science Ehud Shapiro1,2, Tamir Biezuner1,2 and Sten Linnarsson3 Abstract | The unabated progress in next-generation sequencing technologies is fostering a wave of new genomics, epigenomics, transcriptomics and proteomics technologies. These sequencing-based technologies are increasingly being targeted to individual cells, which will allow many new and longstanding questions to be addressed. For example, single-cell genomics will help to uncover cell lineage relationships; single-cell transcriptomics will supplant the coarse notion of marker-based cell types; and single-cell epigenomics and proteomics will allow the functional states of individual cells to be analysed. These technologies will become integrated within a decade or so, enabling high-throughput, multi-dimensional analyses of individual cells that will produce detailed knowledge of the cell lineage trees of higher organisms, including humans. Such studies will have important implications for both basic biological research and medicine. Next-generation sequencing DNA sequencing has undergone constant improvement Single cells can be studied and tracked using many (NGS). High-throughput DNA since its inception in the 1970s. Today, next-generation detection technologies, including quantitative imaging sequencing of a large number sequencing (NGS) approaches are accelerating in speed and mass spectrometry. However, our Review focuses on of DNA molecules in parallel. and decreasing in cost more quickly than Moore’s law1. single-cell analysis using DNA-sequencing-based tech- There is a trade-off between read length and throughput DNA sequencing technologies have improved in preci- nologies. Although single-cell sequencing-based analysis 14 that depends on the sion and throughput, and have enabled the sequenc- has been applied to both unicellular and multicellular sequencing technology, ing of entire genomes of species2,3 and individuals4. organisms15, this Review focuses on mammalian (pri- run time and quality. An increasing number of questions can be addressed marily mouse and human) single-cell analysis. We first by DNA-sequencing-based technologies. In particular, survey current technologies for single-cell isolation, transcriptomic5, epigenomic6 and proteomic7 analyses which is essential for DNA-sequencing-based single- are being carried out using methods that reduce a spe- cell analysis. We then review technologies for single-cell cific analysis problem to a DNA-sequencing problem, as genomic and transcriptomic analysis, and their applica- explained in FIG. 1. tions. We briefly discuss methods for sequencing-based 1Department of Computer DNA sequencing technology has not only scaled up epigenomic and proteomic analyses that have yet to be Science and Applied Math rapidly in throughput but — through advances in sam- scaled to single cells. Finally, we describe the impact that and 2Department of Biological Chemistry, Weizmann Institute ple preparation — has also scaled down in terms of the the integration of these methods will have on whole- of Science, Rehovot 76100, amount of DNA that is required for analysis, to the point organism science (FIG. 1). We predict an era of integrated Israel. at which it is now feasible to analyse the DNA content of single-cell genomic, epigenomic, transcriptomic and 3Department of Medical individual cells8,9. This opens up a wealth of previously proteomic analysis, which we believe will revolutionize Biochemistry and Biophysics, Karolinska Institutet, Scheeles impossible applications in both basic research and clini- whole-organism science by enabling the reconstruction väg 2, 17177 Stockholm, cal science. Examples are: the study of microorganisms of organismal cell lineage trees for higher organisms, cul- Sweden. that cannot be cultured, using direct single-cell genome minating in the reconstruction of an entire human cell Correspondence to E.S. and S.L. sequencing10; transcriptome analysis of rare, circulating lineage tree16, which will have broad implications for e-mails: ehud.shapiro@ tumour cells11; characterization of the earliest differentia- human biology and medicine. weizmann.ac.il; [email protected] tion events in human embryogenesis; the investigation of Naturally, in such a diverse, rapidly developing and doi:10.1038/nrg3542 transcriptional noise and stochastic fate choice; and the interdisciplinary field, we cannot possibly cover all of the Published online 30 July 2013 study of tumour heterogeneity12 and microevolution13. work that has been carried out over the past few years. 618 | SEPTEMBER 2013 | VOLUME 14 www.nature.com/reviews/genetics © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS a Cell Assay that transforms a DNA library Sequence data Computational Knowledge of cell property into a DNA analysis inferring the cell property library reflecting it DNA sequencer GATCGATCATTGCTAGCTC the cell property (e.g. transcriptome) TACGTAGCTAGCTAGCTAG CATAGCTAGCCATAGCTTA mRNA ATCGCTAGCTATTCAGCTC miRNA b Assays that transform each DNA libraries Sequence data Computational Knowledge of P1 cell property Pi to a DNA analysis inferring (e.g. mutations) GATCGATCATTGCTAGCTC library reflecting Pi properties P1 … Pn TGGCATGA TACGTAGCTAGCTAGCTAG ATGTCTTA CATAGCTAGCCATAGCTTA GGAGATTG Cell ATCGCTAGCTATTCAGCTC Knowledge of P DNA sequencer GCTAGCTATAGCTCTAGCT 2 AGCATTCGATCTAGCTATG (e.g. methylation) TTGCTATGCTATCGACTAG Me … … CTAGCTATCGCTCTACGAC Me TGACTGCTTAGCTATTCAG CTC GCTAGCTATAGCTCT Knowledge of Pn AGCATTCGATCTAGCTATG (e.g. mRNA count) CTGCTATGCTATCAGCGAT mRNA miRNA Figure 1 | Single-cell sequencing-based analysis methods and their anticipated integration. a | Architecture of Nature Reviews | Genetics single-cell DNA-sequencing-based technologies. Current implementations include single-cell genomics (targeted exome or mutational analysis9,16,56,59, copy-number variation8,9,58, and recombination analysis in germ cells27,68), transcriptomics (transcriptome analysis11,99,102,104 and recombination analysis in the immune system136) and epigenomics113. b | Architecture of future integrated single-cell DNA-sequencing-based analysis. We expect that within a decade this architecture will allow the simultaneous analysis of multiple properties of an individual cell, including genomics3,4,38,60–63,76,78–81,83,84,137–139, epigenomics (methylation6,82,106,107,140, chromatin108 and conformational110,111 analysis), transcriptomics (transcriptome analysis5,141–143, allele-specific gene expression94 and molecule counting93–97) and Organismal cell lineage tree proteomics7,127, all of which are currently limited to bulk experiments. A mathematical entity capturing all cell division and death events in the life of an organism up to a particular time point. The tree consists of Also, we expect that by the time this Review is published, Their disadvantages are that they are only applicable labelled nodes, which represent all organismal cells, and additional progress will have been made, which we to cells in suspension, they are low-throughput, and directed edges, which represent have been unable to cover. We apologize to the authors they are susceptible to errors, such as misidentifica- progeny relationships among whose work we have not discussed. tion of a cell under a microscope. These disadvantages them. A reconstructed tree are partially addressed by semi-automated devices for describes lineage relationships Methods for single-cell isolation cell isolation, with which an expert operator can iso- among cells sampled from an 20 organism, and is precise only Tissues are rarely homogenous, and typically consist of late approximately 50–100 cells per hour . A different if it is a subtree of the (true) tens or hundreds of distinct cell types, which are often approach, which is also classified as micromanipula- organismal cell lineage tree. intermingled and present at widely different abun- tion, is the optical tweezers technology, which uses a dances. Single cells can be isolated from such tissues in laser beam to capture cells. Although not commonly Cell type (TABLE 1) A classification of cells by various ways , which can be classified as either used, it allows specific cell micromanipulation and 21 morphology, genotype, unbiased (randomized) or biased (targeted) sampling. measurement . phenotype or developmental In principle, an unbiased sample better reflects the Cell isolation can also be achieved by flow sorting origin. There is no consensus composition of the tissue, but a targeted sample may using fluorescence-activated cell sorting (FACS), either on which properties are be necessary in order to isolate rare cell types. using cell-type-specific markers for a biased, targeted necessary and sufficient for this classification, nor is there There are two key steps in the isolation of single sample, and/or using the light-scattering properties of general agreement on the cells from a solid tissue. First, the tissue must be cells to obtain an unbiased sample. The main advan- actual number of cell types or removed from the animal or plant — typically by dis- tages of FACS-based sorting are the ability to choose their proper classification in section or biopsy — and dissociated into its constituent between biased and unbiased isolation, high levels of any higher organism, including 12 in humans. individual cells, usually using enzymatic disaggrega- accuracy and high-throughput single-cell isolation . tion. Second,
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