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Journal ArticleDOI

In situ sequencing for RNA analysis in preserved tissue and cells

TL;DR: In situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections is demonstrated and the method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue is developed.
Abstract: Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.
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Journal ArticleDOI
Aviv Regev1, Aviv Regev2, Aviv Regev3, Sarah A. Teichmann4, Sarah A. Teichmann5, Sarah A. Teichmann6, Eric S. Lander2, Eric S. Lander1, Eric S. Lander7, Ido Amit8, Christophe Benoist7, Ewan Birney4, Bernd Bodenmiller9, Bernd Bodenmiller4, Peter J. Campbell5, Peter J. Campbell6, Piero Carninci5, Menna R. Clatworthy10, Hans Clevers11, Bart Deplancke12, Ian Dunham4, James Eberwine13, Roland Eils14, Roland Eils15, Wolfgang Enard16, Andrew Farmer, Lars Fugger17, Berthold Göttgens5, Nir Hacohen7, Nir Hacohen2, Muzlifah Haniffa18, Martin Hemberg6, Seung K. Kim19, Paul Klenerman17, Paul Klenerman20, Arnold R. Kriegstein21, Ed S. Lein22, Sten Linnarsson23, Emma Lundberg24, Emma Lundberg19, Joakim Lundeberg24, Partha P. Majumder, John C. Marioni4, John C. Marioni5, John C. Marioni6, Miriam Merad25, Musa M. Mhlanga26, Martijn C. Nawijn27, Mihai G. Netea28, Garry P. Nolan19, Dana Pe'er29, Anthony Phillipakis2, Chris P. Ponting30, Stephen R. Quake19, Wolf Reik5, Wolf Reik6, Wolf Reik31, Orit Rozenblatt-Rosen2, Joshua R. Sanes7, Rahul Satija32, Ton N. Schumacher33, Alex K. Shalek2, Alex K. Shalek34, Alex K. Shalek1, Ehud Shapiro8, Padmanee Sharma35, Jay W. Shin, Oliver Stegle4, Michael R. Stratton6, Michael J. T. Stubbington6, Fabian J. Theis36, Matthias Uhlen24, Matthias Uhlen37, Alexander van Oudenaarden11, Allon Wagner38, Fiona M. Watt39, Jonathan S. Weissman, Barbara J. Wold40, Ramnik J. Xavier, Nir Yosef38, Nir Yosef34, Human Cell Atlas Meeting Participants 
05 Dec 2017-eLife
TL;DR: An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease.
Abstract: The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

1,391 citations


Cites background or methods from "In situ sequencing for RNA analysis..."

  • ...Finally, more recent studies have performed RNA-seq in situ in cells and in preserved tissue sections (Ke et al., 2013; Lee et al., 2014)....

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  • ...Techniques are being developed to assay: in situ gene expression in tissues at single-cell and even sub-cellular resolution (Chen et al., 2015b; Ke et al., 2013; Lee et al., 2014; Lubeck et al., 2014; Shah et al., 2016; Ståhl et al., 2016); the distribution of scores of proteins at cellular or…...

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Journal ArticleDOI
22 Feb 2018-Cell
TL;DR: This study developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices, and built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression.

1,234 citations


Cites background from "In situ sequencing for RNA analysis..."

  • ...…2015; Lorthongpanich et al., 2013; Mooijman et al., 2016; Nagano et al., 2013; Smallwood et al., 2014), and in situ analysis (Chen et al., 2015; Ke et al., 2013; Lee et al., Cell 172, 1091–1107, February 22, 2018 ª 2018 Elsevier Inc. 1091 2014), may provide further information regarding…...

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Journal ArticleDOI
TL;DR: This review provides a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades including applications in bioanalysis, diagnostics, nanotechnology, materials science, and device integration.
Abstract: Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

1,144 citations

Journal ArticleDOI
TL;DR: The first, to the authors' knowledge, single cell whole transcriptome analysis of human adult cortical samples is described, establishing an experimental and analytical framework with which the complexity of the human brain can be dissected on the single cell level.
Abstract: The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

1,121 citations

Journal ArticleDOI
TL;DR: Recently developed single-cell mRNA-sequencing methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells, which provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells.

987 citations


Cites methods from "In situ sequencing for RNA analysis..."

  • ...These methods, including FISSEQ (Lee et al., 2014a; Mitra et al., 2003), TIVA (Lovatt et al., 2014), and padlock probes based methods (Ke et al., 2013), have low sensitivity, which comes at the cost of obtaining spatial information (Crosetto et al., 2015)....

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References
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Journal ArticleDOI
TL;DR: The RNA-Seq approach to transcriptome profiling that uses deep-sequencing technologies provides a far more precise measurement of levels of transcripts and their isoforms than other methods.
Abstract: RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

11,528 citations

Journal ArticleDOI
27 Nov 2008-Nature
TL;DR: An in-depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments yielding a digital inventory of gene and mRNA isoform expression suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.
Abstract: Through alternative processing of pre-messenger RNAs, individual mammalian genes often produce multiple mRNA and protein isoforms that may have related, distinct or even opposing functions. Here we report an in-depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments, yielding a digital inventory of gene and mRNA isoform expression. Analyses in which sequence reads are mapped to exon-exon junctions indicated that 92-94% of human genes undergo alternative splicing, 86% with a minor isoform frequency of 15% or more. Differences in isoform-specific read densities indicated that most alternative splicing and alternative cleavage and polyadenylation events vary between tissues, whereas variation between individuals was approximately twofold to threefold less common. Extreme or 'switch-like' regulation of splicing between tissues was associated with increased sequence conservation in regulatory regions and with generation of full-length open reading frames. Patterns of alternative splicing and alternative cleavage and polyadenylation were strongly correlated across tissues, suggesting coordinated regulation of these processes, and sequence conservation of a subset of known regulatory motifs in both alternative introns and 3' untranslated regions suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.

4,711 citations

Journal ArticleDOI
TL;DR: An automatic subpixel registration algorithm that minimizes the mean square intensity difference between a reference and a test data set, which can be either images (two-dimensional) or volumes (three-dimensional).
Abstract: We present an automatic subpixel registration algorithm that minimizes the mean square intensity difference between a reference and a test data set, which can be either images (two-dimensional) or volumes (three-dimensional). It uses an explicit spline representation of the images in conjunction with spline processing, and is based on a coarse-to-fine iterative strategy (pyramid approach). The minimization is performed according to a new variation (ML*) of the Marquardt-Levenberg algorithm for nonlinear least-square optimization. The geometric deformation model is a global three-dimensional (3-D) affine transformation that can be optionally restricted to rigid-body motion (rotation and translation), combined with isometric scaling. It also includes an optional adjustment of image contrast differences. We obtain excellent results for the registration of intramodality positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) data. We conclude that the multiresolution refinement strategy is more robust than a comparable single-stage method, being less likely to be trapped into a false local optimum. In addition, our improved version of the Marquardt-Levenberg algorithm is faster.

2,801 citations

Journal ArticleDOI
TL;DR: A single-cell digital gene expression profiling assay with only a single mouse blastomere is described, which detected the expression of 75% more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads.
Abstract: Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.

2,659 citations

Journal ArticleDOI
07 Apr 2011-Nature
TL;DR: It is shown that with flow-sorted nuclei, whole genome amplification and next generation sequencing the authors can accurately quantify genomic copy number within an individual nucleus and indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.
Abstract: Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse 'pseudodiploid' cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.

2,426 citations

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