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1.
Science ; 367(6482): 1151-1156, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32139547

RESUMEN

The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics.


Asunto(s)
Estabilidad del ARN , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcripción Genética , Animales , Humanos , Indicadores y Reactivos/química , Células K562 , Ratones , Uridina/análogos & derivados
3.
Nat Commun ; 11(1): 89, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31900397

RESUMEN

RNA sequencing experiments generate large amounts of information about expression levels of genes. Although they are mainly used for quantifying expression levels, they contain much more biologically important information such as copy number variants (CNVs). Here, we present CaSpER, a signal processing approach for identification, visualization, and integrative analysis of focal and large-scale CNV events in multiscale resolution using either bulk or single-cell RNA sequencing data. CaSpER integrates the multiscale smoothing of expression signal and allelic shift signals for CNV calling. The allelic shift signal measures the loss-of-heterozygosity (LOH) which is valuable for CNV identification. CaSpER employs an efficient methodology for the generation of a genome-wide B-allele frequency (BAF) signal profile from the reads and utilizes it for correction of CNVs calls. CaSpER increases the utility of RNA-sequencing datasets and complements other tools for complete characterization and visualization of the genomic and transcriptomic landscape of single cell and bulk RNA sequencing data.


Asunto(s)
Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Programas Informáticos , Algoritmos , Alelos , Genómica , Humanos , Pérdida de Heterocigocidad , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ARN , Análisis de la Célula Individual
4.
Chem Commun (Camb) ; 56(16): 2423-2426, 2020 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-31994543

RESUMEN

A highly sensitive laser-induced fluorescence detection system with real-time imaging focusing instead of the use of fluorescent reagents were developed for the detection of analytes in nanocapillaries. A cylindrical lens was adopted for the shaping of a laser beam to increase its spatial resolution. The limit of detection with a capillary having a radius of 440 nm was 6.76 yoctomoles (or four molecules) for fluorescein, and 84.5 yoctomoles (or 51 molecules) for FAM-labeled Hsa-miR-17. The designed system exhibited high sensitivity and stability for femtoliters of sample and was hence indicated to be suitable for analyses of single cells.


Asunto(s)
Fluorescencia , Colorantes Fluorescentes/química , Rayos Láser , MicroARNs/análisis , Imagen Óptica , Análisis de la Célula Individual , Humanos , Espectrometría de Fluorescencia , Factores de Tiempo
5.
Chem Commun (Camb) ; 56(10): 1561-1564, 2020 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-31930270

RESUMEN

A glass nanopipette functionalized with ATP-responsive gold nanoparticle assemblies was developed for ATP detection in single-cells and used for analysing the content change of ATP during electrostimulus (ES)-induced apoptosis. The variation of ATP content in single normal cells and cancer cells during apoptosis was detected by the method.


Asunto(s)
Adenosina Trifosfato/análisis , Oro/química , Nanopartículas del Metal/química , Nanotecnología/métodos , Apoptosis , Línea Celular , Electricidad , Vidrio/química , Células HeLa , Humanos , Microscopía Fluorescente , Nanoporos , Análisis de la Célula Individual
6.
Genes Dev ; 34(3-4): 239-249, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31919193

RESUMEN

Addressing the complexity of organogenesis at a system-wide level requires a complete understanding of adult cell types, their origin, and precursor relationships. The Drosophila ovary has been a model to study how coordinated stem cell units, germline, and somatic follicle stem cells maintain and renew an organ. However, lack of cell type-specific tools have limited our ability to study the origin of individual cell types and stem cell units. Here, we used a single-cell RNA sequencing approach to uncover all known cell types of the developing ovary, reveal transcriptional signatures, and identify cell type-specific markers for lineage tracing. Our study identifies a novel cell type corresponding to the elusive follicle stem cell precursors and predicts subtypes of known cell types. Altogether, we reveal a previously unanticipated complexity of the developing ovary and provide a comprehensive resource for the systematic analysis of ovary morphogenesis.


Asunto(s)
Drosophila/citología , Folículo Ovárico/citología , Células Madre/citología , Animales , Drosophila/genética , Drosophila/metabolismo , Femenino , Modelos Animales , Ovario/citología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcripción Genética
7.
Genome Biol ; 21(1): 10, 2020 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-31937348

RESUMEN

Although scRNA-seq is now ubiquitously adopted in studies of intratumor heterogeneity, detection of somatic mutations and inference of clonal membership from scRNA-seq is currently unreliable. We propose DENDRO, an analysis method for scRNA-seq data that clusters single cells into genetically distinct subclones and reconstructs the phylogenetic tree relating the subclones. DENDRO utilizes transcribed point mutations and accounts for technical noise and expression stochasticity. We benchmark DENDRO and demonstrate its application on simulation data and real data from three cancer types. In particular, on a mouse melanoma model in response to immunotherapy, DENDRO delineates the role of neoantigens in treatment response.


Asunto(s)
Heterogeneidad Genética , Técnicas Genéticas , Neoplasias/genética , Filogenia , Programas Informáticos , Animales , Humanos , Ratones , Análisis de la Célula Individual
8.
J Forensic Sci ; 65(1): 295-303, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30859587

RESUMEN

A set of historic murders, known as the "Jack the Ripper murders," started in London in August 1888. The killer's identity has remained a mystery to date. Here, we describe the investigation of, to our knowledge, the only remaining physical evidence linked to these murders, recovered from one of the victims at the scene of the crime. We applied novel, minimally destructive techniques for sample recovery from forensically relevant stains on the evidence and separated single cells linked to the suspect, followed by phenotypic analysis. The mtDNA profiles of both the victim and the suspect matched the corresponding reference samples, fortifying the link of the evidence to the crime scene. Genomic DNA from single cells recovered from the evidence was amplified, and the phenotypic information acquired matched the only witness statement regarded as reliable. To our knowledge, this is the most advanced study to date regarding this case.


Asunto(s)
Vestuario , Dermatoglifia del ADN/métodos , Genética Forense/métodos , Homicidio/historia , Manchas de Sangre , Vestuario/historia , Víctimas de Crimen , Criminales , ADN Mitocondrial/genética , ADN Mitocondrial/aislamiento & purificación , Fluorescencia , Historia del Siglo XIX , Humanos , Rayos Infrarrojos , Captura por Microdisección con Láser , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Análisis de la Célula Individual , Reino Unido , Secuenciación Completa del Genoma
9.
Nucleic Acids Res ; 48(3): 1146-1163, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-31853540

RESUMEN

Long Interspersed Element-1 (LINE-1) retrotransposition contributes to inter- and intra-individual genetic variation and occasionally can lead to human genetic disorders. Various strategies have been developed to identify human-specific LINE-1 (L1Hs) insertions from short-read whole genome sequencing (WGS) data; however, they have limitations in detecting insertions in complex repetitive genomic regions. Here, we developed a computational tool (PALMER) and used it to identify 203 non-reference L1Hs insertions in the NA12878 benchmark genome. Using PacBio long-read sequencing data, we identified L1Hs insertions that were absent in previous short-read studies (90/203). Approximately 81% (73/90) of the L1Hs insertions reside within endogenous LINE-1 sequences in the reference assembly and the analysis of unique breakpoint junction sequences revealed 63% (57/90) of these L1Hs insertions could be genotyped in 1000 Genomes Project sequences. Moreover, we observed that amplification biases encountered in single-cell WGS experiments led to a wide variation in L1Hs insertion detection rates between four individual NA12878 cells; under-amplification limited detection to 32% (65/203) of insertions, whereas over-amplification increased false positive calls. In sum, these data indicate that L1Hs insertions are often missed using standard short-read sequencing approaches and long-read sequencing approaches can significantly improve the detection of L1Hs insertions present in individual genomes.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Análisis de Secuencia de ADN/métodos , Línea Celular , Genoma Humano , Humanos , Polimorfismo Genético , Análisis de la Célula Individual , Programas Informáticos , Secuenciación Completa del Genoma
10.
Recent Results Cancer Res ; 215: 89-104, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31605225

RESUMEN

Circulating tumor cells (CTCs) represent novel biomarkers, since they are obtainable through a simple and noninvasive blood draw or liquid biopsy. Here, we review the high-definition single-cell analysis (HD-SCA) workflow, which brings together modern methods of immunofluorescence with more sophisticated image processing to rapidly and accurately detect rare tumor cells among the milieu of platelets, erythrocytes, and leukocytes in the peripheral blood. In particular, we discuss progress in methods to measure CTC morphology and subcellular protein expression, and we highlight some initial applications that lead to fundamental new insights about the hematogenous phase of cancer, as well as its performance in early-stage diagnosis and treatment monitoring. We end with an outlook on how to further probe CTCs and the unique advantages of the HD-SCA workflow for improving the precision of cancer care.


Asunto(s)
Biología Computacional , Neoplasias/patología , Células Neoplásicas Circulantes/metabolismo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias/diagnóstico , Neoplasias/terapia , Células Neoplásicas Circulantes/patología , Análisis de la Célula Individual
11.
Talanta ; 206: 120174, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31514890

RESUMEN

A method of simultaneous cell counting and determination of metals in single cells using time-resolved inductively coupled plasma-mass spectrometry (ICP-MS) was reported. A facile, low cost and highly efficient single-cell introduction system of time-resolved ICP-MS consists of a flow cell, a visual contrast calibration device, a customized nebulizer and a fabricated spray chamber. The flow cell includes a cell sample tube, a sheath liquid tube and a flow chamber. The visual contrast calibration device was composed of a microscope with a 16 × microscope objective (160 × total magnification). The flow chamber was used to combine a flow of red blood cell suspension (0.800 µL/min) and a flow of PBS (4.40 µL/min) into the nebulizer. The intact cells were directly introduced with the single-cell introduction system into the plasma via nebulizing, and then ion plumes corresponding to single cells were individually detected with mass spectrometer. The frequency of the spikes directly reflects the number of cells, and the intensity of spikes is proportional to the concentration of copper within one cell. The single-cell introduction system can be transported into the ICP-MS via a customized transport system with 100% efficiency. A high cell introduction efficiency into the plasma supports for a reduction of cell consumption. The Cu signal frequency was about 120 cell events per minute. This single-cell introduction system simplifies the introduction of individual and intact cells. The copper content in single red blood cell was 0.20-0.40 fg.


Asunto(s)
Cobre/análisis , Eritrocitos/química , Humanos , Límite de Detección , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Nebulizadores y Vaporizadores , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodos
14.
Nat Biotechnol ; 37(12): 1482-1492, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31796933

RESUMEN

The high-dimensional data created by high-throughput technologies require visualization tools that reveal data structure and patterns in an intuitive form. We present PHATE, a visualization method that captures both local and global nonlinear structure using an information-geometric distance between data points. We compare PHATE to other tools on a variety of artificial and biological datasets, and find that it consistently preserves a range of patterns in data, including continual progressions, branches and clusters, better than other tools. We define a manifold preservation metric, which we call denoised embedding manifold preservation (DEMaP), and show that PHATE produces lower-dimensional embeddings that are quantitatively better denoised as compared to existing visualization methods. An analysis of a newly generated single-cell RNA sequencing dataset on human germ-layer differentiation demonstrates how PHATE reveals unique biological insight into the main developmental branches, including identification of three previously undescribed subpopulations. We also show that PHATE is applicable to a wide variety of data types, including mass cytometry, single-cell RNA sequencing, Hi-C and gut microbiome data.


Asunto(s)
Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Procesamiento de Imagen Asistida por Computador/métodos , Algoritmos , Animales , Macrodatos , Diferenciación Celular , Células Cultivadas , Simulación por Computador , Bases de Datos Genéticas , Microbioma Gastrointestinal , Humanos , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula Individual
16.
Nat Biotechnol ; 37(12): 1458-1465, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31792411

RESUMEN

Identifying the causes of human diseases requires deconvolution of abnormal molecular phenotypes spanning DNA accessibility, gene expression and protein abundance1-3. We present a single-cell framework that integrates highly multiplexed protein quantification, transcriptome profiling and analysis of chromatin accessibility. Using this approach, we establish a normal epigenetic baseline for healthy blood development, which we then use to deconvolve aberrant molecular features within blood from patients with mixed-phenotype acute leukemia4,5. Despite widespread epigenetic heterogeneity within the patient cohort, we observe common malignant signatures across patients as well as patient-specific regulatory features that are shared across phenotypic compartments of individual patients. Integrative analysis of transcriptomic and chromatin-accessibility maps identified 91,601 putative peak-to-gene linkages and transcription factors that regulate leukemia-specific genes, such as RUNX1-linked regulatory elements proximal to the marker gene CD69. These results demonstrate how integrative, multiomic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples.


Asunto(s)
Cromatina/genética , Leucemia Bifenotípica Aguda/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Células de la Médula Ósea/citología , Cromatina/química , Análisis por Conglomerados , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Epigénesis Genética/genética , Epigenómica/métodos , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Secuencias Reguladoras de Ácidos Nucleicos/genética
17.
Nat Med ; 25(12): 1894-1904, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31792459

RESUMEN

How obesity and elevated androgen levels in women with polycystic ovary syndrome (PCOS) affect their offspring is unclear. In a Swedish nationwide register-based cohort and a clinical case-control study from Chile, we found that daughters of mothers with PCOS were more likely to be diagnosed with PCOS. Furthermore, female mice (F0) with PCOS-like traits induced by late-gestation injection of dihydrotestosterone, with and without obesity, produced female F1-F3 offspring with PCOS-like reproductive and metabolic phenotypes. Sequencing of single metaphase II oocytes from F1-F3 offspring revealed common and unique altered gene expression across all generations. Notably, four genes were also differentially expressed in serum samples from daughters in the case-control study and unrelated women with PCOS. Our findings provide evidence of transgenerational effects in female offspring of mothers with PCOS and identify possible candidate genes for the prediction of a PCOS phenotype in future generations.


Asunto(s)
Andrógenos/metabolismo , Oocitos/metabolismo , Síndrome del Ovario Poliquístico/genética , Efectos Tardíos de la Exposición Prenatal/genética , Animales , Estudios de Cohortes , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Núcleo Familiar , /metabolismo , Oocitos/inmunología , Fenotipo , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/diagnóstico , Síndrome del Ovario Poliquístico/fisiopatología , Embarazo , Efectos Tardíos de la Exposición Prenatal/diagnóstico , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Análisis de la Célula Individual
18.
Nat Genet ; 51(12): 1714-1722, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31784732

RESUMEN

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.


Asunto(s)
Histonas/metabolismo , Rabdomiosarcoma/genética , Factores de Transcripción/genética , Acetilación , Benzamidas/farmacología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Elementos de Facilitación Genéticos , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/genética , Humanos , Piridinas/farmacología , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Estabilidad del ARN , Factores de Transcripción SOXE/genética , Análisis de la Célula Individual
19.
BMC Bioinformatics ; 20(Suppl 24): 672, 2019 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-31861972

RESUMEN

BACKGROUND: Various statistical models have been developed to model the single cell RNA-seq expression profiles, capture its multimodality, and conduct differential gene expression test. However, for expression data generated by different experimental design and platforms, there is currently lack of capability to determine the most proper statistical model. RESULTS: We developed an R package, namely Multi-Modal Model Selection (M3S), for gene-wise selection of the most proper multi-modality statistical model and downstream analysis, useful in a single-cell or large scale bulk tissue transcriptomic data. M3S is featured with (1) gene-wise selection of the most parsimonious model among 11 most commonly utilized ones, that can best fit the expression distribution of the gene, (2) parameter estimation of a selected model, and (3) differential gene expression test based on the selected model. CONCLUSION: A comprehensive evaluation suggested that M3S can accurately capture the multimodality on simulated and real single cell data. An open source package and is available through GitHub at https://github.com/zy26/M3S.


Asunto(s)
ARN/genética , Análisis de Secuencia de ARN , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Genéticos , Análisis de la Célula Individual , Transcriptoma
20.
Genome Biol ; 20(1): 290, 2019 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-31856883

RESUMEN

A variety of methods have been developed to demultiplex pooled samples in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or sample genotypes prior to pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled samples. scSplit also enables mapping clusters to original samples. Using simulated, merged, and pooled multi-individual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to samples without external genotype information and is available at: https://github.com/jon-xu/scSplit.


Asunto(s)
Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Humanos
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