RESUMEN
Many datasets are being produced by consortia that seek to characterize healthy and disease tissues at single-cell resolution. While biospecimen and experimental information is often captured, detailed metadata standards related to data matrices and analysis workflows are currently lacking. To address this, we develop the matrix and analysis metadata standards (MAMS) to serve as a resource for data centers, repositories, and tool developers. We define metadata fields for matrices and parameters commonly utilized in analytical workflows and developed the rmams package to extract MAMS from single-cell objects. Overall, MAMS promotes the harmonization, integration, and reproducibility of single-cell data across platforms.
Asunto(s)
Metadatos , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/normas , Reproducibilidad de los Resultados , Humanos , Programas InformáticosRESUMEN
Gene set scoring (GSS) has been routinely conducted for gene expression analysis of bulk or single-cell RNA sequencing (RNA-seq) data, which helps to decipher single-cell heterogeneity and cell type-specific variability by incorporating prior knowledge from functional gene sets. Single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) is a powerful technique for interrogating single-cell chromatin-based gene regulation, and genes or gene sets with dynamic regulatory potentials can be regarded as cell type-specific markers as if in single-cell RNA-seq (scRNA-seq). However, there are few GSS tools specifically designed for scATAC-seq, and the applicability and performance of RNA-seq GSS tools on scATAC-seq data remain to be investigated. Here, we systematically benchmarked ten GSS tools, including four bulk RNA-seq tools, five scRNA-seq tools, and one scATAC-seq method. First, using matched scATAC-seq and scRNA-seq datasets, we found that the performance of GSS tools on scATAC-seq data was comparable to that on scRNA-seq, suggesting their applicability to scATAC-seq. Then, the performance of different GSS tools was extensively evaluated using up to ten scATAC-seq datasets. Moreover, we evaluated the impact of gene activity conversion, dropout imputation, and gene set collections on the results of GSS. Results show that dropout imputation can significantly promote the performance of almost all GSS tools, while the impact of gene activity conversion methods or gene set collections on GSS performance is more dependent on GSS tools or datasets. Finally, we provided practical guidelines for choosing appropriate preprocessing methods and GSS tools in different application scenarios.
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Algoritmos , Benchmarking , Secuenciación de Inmunoprecipitación de Cromatina , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/normas , Humanos , Secuenciación de Inmunoprecipitación de Cromatina/métodos , RNA-Seq/métodos , RNA-Seq/normas , Análisis de Secuencia de ARN/métodos , Análisis de Secuencia de ARN/normas , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Cromatina/genética , Cromatina/metabolismoRESUMEN
Considerable effort has been devoted to refining experimental protocols to reduce levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. To evaluate the effect of equalization on various protocols, we developed Scaffold, a simulation framework that models each step of an scRNA-seq experiment. Numerical experiments demonstrate that equalization reduces variation in sequencing depth and gene-specific expression variability. We then performed a set of experiments in vitro with and without the equalization step and found that equalization increases the number of genes that are detected in every cell by 17-31%, improves discovery of biologically relevant genes, and reduces nuisance signals associated with cell cycle. Further support is provided in an analysis of publicly available data.
Asunto(s)
Biblioteca de Genes , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Biología Computacional/métodos , Bases de Datos Genéticas , Perfilación de la Expresión Génica/métodos , Humanos , RNA-Seq/normas , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/normas , Programas InformáticosRESUMEN
With the proliferating studies of human cancers by single-cell RNA sequencing technique (scRNA-seq), cellular heterogeneity, immune landscape and pathogenesis within diverse cancers have been uncovered successively. The exponential explosion of massive cancer scRNA-seq datasets in the past decade are calling for a burning demand to be integrated and processed for essential investigations in tumor microenvironment of various cancer types. To fill this gap, we developed a database of Cancer Single-cell Expression Map (CancerSCEM, https://ngdc.cncb.ac.cn/cancerscem), particularly focusing on a variety of human cancers. To date, CancerSCE version 1.0 consists of 208 cancer samples across 28 studies and 20 human cancer types. A series of uniformly and multiscale analyses for each sample were performed, including accurate cell type annotation, functional gene expressions, cell interaction network, survival analysis and etc. Plus, we visualized CancerSCEM as a user-friendly web interface for users to browse, search, online analyze and download all the metadata as well as analytical results. More importantly and unprecedentedly, the newly-constructed comprehensive online analyzing platform in CancerSCEM integrates seven analyze functions, where investigators can interactively perform cancer scRNA-seq analyses. In all, CancerSCEM paves an informative and practical way to facilitate human cancer studies, and also provides insights into clinical therapy assessments.
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Bases de Datos Genéticas , Neoplasias/genética , Programas Informáticos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias/clasificación , RNA-Seq , Análisis de la Célula Individual/normas , Microambiente Tumoral/genéticaRESUMEN
Large single-cell atlases are now routinely generated to serve as references for analysis of smaller-scale studies. Yet learning from reference data is complicated by batch effects between datasets, limited availability of computational resources and sharing restrictions on raw data. Here we introduce a deep learning strategy for mapping query datasets on top of a reference called single-cell architectural surgery (scArches). scArches uses transfer learning and parameter optimization to enable efficient, decentralized, iterative reference building and contextualization of new datasets with existing references without sharing raw data. Using examples from mouse brain, pancreas, immune and whole-organism atlases, we show that scArches preserves biological state information while removing batch effects, despite using four orders of magnitude fewer parameters than de novo integration. scArches generalizes to multimodal reference mapping, allowing imputation of missing modalities. Finally, scArches retains coronavirus disease 2019 (COVID-19) disease variation when mapping to a healthy reference, enabling the discovery of disease-specific cell states. scArches will facilitate collaborative projects by enabling iterative construction, updating, sharing and efficient use of reference atlases.
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Conjuntos de Datos como Asunto/normas , Aprendizaje Profundo , Especificidad de Órganos , Análisis de la Célula Individual/normas , Animales , COVID-19/patología , Humanos , Ratones , Estándares de Referencia , SARS-CoV-2/patogenicidadRESUMEN
Compositional changes of cell types are main drivers of biological processes. Their detection through single-cell experiments is difficult due to the compositionality of the data and low sample sizes. We introduce scCODA ( https://github.com/theislab/scCODA ), a Bayesian model addressing these issues enabling the study of complex cell type effects in disease, and other stimuli. scCODA demonstrated excellent detection performance, while reliably controlling for false discoveries, and identified experimentally verified cell type changes that were missed in original analyses.
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Análisis de la Célula Individual/métodos , Teorema de Bayes , Benchmarking , Perfilación de la Expresión Génica , Humanos , Modelos Estadísticos , Tamaño de la Muestra , Análisis de la Célula Individual/normasRESUMEN
Single-cell RNA (scRNA) profiling or scRNA-sequencing (scRNA-seq) makes it possible to parallelly investigate diverse molecular features of multiple types of cells in a given plant tissue and discover cell developmental processes. In this study, we evaluated the effects of sample size (i.e., cell number) on the outcome of single-cell transcriptome analysis by sampling different numbers of cells from a pool of ~57,000 Arabidopsis thaliana root cells integrated from five published studies. Our results indicated that the most significant principal components could be achieved when 20,000-30,000 cells were sampled, a relatively high reliability of cell clustering could be achieved by using ~20,000 cells with little further improvement by using more cells, 96% of the differentially expressed genes could be successfully identified with no more than 20,000 cells, and a relatively stable pseudotime could be estimated in the subsample with 5000 cells. Finally, our results provide a general guide for optimizing sample size to be used in plant scRNA-seq studies.
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Perfilación de la Expresión Génica , ARN de Planta , Análisis de la Célula Individual , Transcriptoma , Arabidopsis/genética , Recuento de Células , Análisis por Conglomerados , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Especificidad de Órganos/genética , Plantas/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/normasRESUMEN
Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.
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Péptidos/aislamiento & purificación , Proteoma/aislamiento & purificación , Proteómica/métodos , Análisis de la Célula Individual/métodos , Animales , Benchmarking , Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Células HeLa , Humanos , Indicadores y Reactivos/química , Ratones , Oocitos/citología , Oocitos/metabolismo , Péptidos/química , Péptidos/clasificación , Cultivo Primario de Células , Proteoma/química , Proteoma/clasificación , Células RAW 264.7 , Análisis de la Célula Individual/normas , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas , Células U937RESUMEN
Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.
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Perfilación de la Expresión Génica/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Transcripción Reversa/genética , Análisis de la Célula Individual/normas , Perfilación de la Expresión Génica/métodos , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de la Célula Individual/métodosRESUMEN
ΩqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision of ΩqPCR were assessed using 800 bp and 1600 bp synthetic telomeres inserted into plasmids, which were measured to be 819 ± 19.6 and 1590 ± 42.3 bp, respectively. This is the first telomere length measuring method verified in this way. The approach uses Ω-probes, a DNA strand containing sequence information that enables: (i) hybridization with the telomere via the 3' and 5' ends that become opposed; (ii) ligation of the hybridized probes to circularize the Ω-probes and (iii) circularized-dependent qPCR due to sequence information for a forward primer, and for a reverse primer binding site, and qPCR hydrolysis probe binding. Read through of the polymerase during qPCR occurs only in circularized Ω-probes, which quantifies their number that is directly proportional to telomere length. When used in concert with information about the cell cycle stage from a single-copy gene, and ploidy, the MTL of single cells measured by ΩqPCR was consistent with that obtained from large sample sizes by TRF.
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Reacción en Cadena de la Polimerasa/métodos , Análisis de la Célula Individual/métodos , Homeostasis del Telómero , Telómero/química , Línea Celular , Humanos , Límite de Detección , Reacción en Cadena de la Polimerasa/normas , Análisis de la Célula Individual/normas , Telómero/genéticaRESUMEN
Advances in whole genome amplification (WGA) techniques enable understanding of the genomic sequence at a single cell level. Demand for single cell dedicated WGA kits (scWGA) has led to the development of several commercial kit. To this point, no robust comparison of all available kits was performed. Here, we benchmark an economical assay, comparing all commercially available scWGA kits. Our comparison is based on targeted sequencing of thousands of genomic loci, including highly mutable regions, from a large cohort of human single cells. Using this approach we have demonstrated the superiority of Ampli1 in genome coverage and of RepliG in reduced error rate. In summary, we show that no single kit is optimal across all categories, highlighting the need for a dedicated kit selection in accordance with experimental requirements.
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Análisis de la Célula Individual/métodos , Secuenciación Completa del Genoma/métodos , Células Cultivadas , Humanos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Sensibilidad y Especificidad , Análisis de la Célula Individual/normas , Secuenciación Completa del Genoma/normasRESUMEN
Cell fate conversion by overexpressing defined factors is a powerful tool in regenerative medicine. However, identifying key factors for cell fate conversion requires laborious experimental efforts; thus, many of such conversions have not been achieved yet. Nevertheless, cell fate conversions found in many published studies were incomplete as the expression of important gene sets could not be manipulated thoroughly. Therefore, the identification of master transcription factors for complete and efficient conversion is crucial to render this technology more applicable clinically. In the past decade, systematic analyses on various single-cell and bulk OMICs data have uncovered numerous gene regulatory mechanisms, and made it possible to predict master gene regulators during cell fate conversion. By virtue of the sparse structure of master transcription factors and the group structure of their simultaneous regulatory effects on the cell fate conversion process, this study introduces a novel computational method predicting master transcription factors based on group sparse optimization technique integrating data from multi-OMICs levels, which can be applicable to both single-cell and bulk OMICs data with a high tolerance of data sparsity. When it is compared with current prediction methods by cross-referencing published and validated master transcription factors, it possesses superior performance. In short, this method facilitates fast identification of key regulators, give raise to the possibility of higher successful conversion rate and in the hope of reducing experimental cost.
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Biología Computacional/métodos , Genómica/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Animales , Sitios de Unión , Linaje de la Célula/genética , Fenómenos Fisiológicos Celulares/genética , Secuenciación de Inmunoprecipitación de Cromatina , Biología Computacional/normas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genómica/normas , Humanos , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Análisis de la Célula Individual/normas , Factores de Transcripción/metabolismo , Transcriptoma , Flujo de TrabajoRESUMEN
Multiplex imaging technologies are now routinely capable of measuring more than 40 antibody-labeled parameters in single cells. However, lateral spillage of signals in densely packed tissues presents an obstacle to the assignment of high-dimensional spatial features to individual cells for accurate cell-type annotation. We devised a method to correct for lateral spillage of cell surface markers between adjacent cells termed REinforcement Dynamic Spillover EliminAtion (REDSEA). The use of REDSEA decreased contaminating signals from neighboring cells. It improved the recovery of marker signals across both isotopic (i.e., Multiplexed Ion Beam Imaging) and immunofluorescent (i.e., Cyclic Immunofluorescence) multiplexed images resulting in a marked improvement in cell-type classification.
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Biomarcadores , Linaje de la Célula , Imagen Molecular/métodos , Animales , Técnica del Anticuerpo Fluorescente/métodos , Procesamiento de Imagen Asistido por Computador , Imagen Molecular/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Relación Señal-Ruido , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/normasAsunto(s)
Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/normas , Análisis de la Célula Individual/métodos , Transcriptoma , Linaje de la Célula/genética , Conjuntos de Datos como Asunto , Humanos , Metadatos , Reproducibilidad de los Resultados , Análisis de la Célula Individual/normasRESUMEN
The transcriptomic diversity of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Unsupervised clustering of SC transcriptomes, which is the default technique for defining cell types, is prone to group cells by technical, rather than biological, variation. Compared to de-novo (unsupervised) clustering, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects and data quality artifacts. Here, we present RCA2, the first algorithm to combine reference projection (batch effect robustness) with graph-based clustering (scalability). In addition, RCA2 provides a user-friendly framework incorporating multiple commonly used downstream analysis modules. RCA2 also provides new reference panels for human and mouse and supports generation of custom panels. Furthermore, RCA2 facilitates cell type-specific QC, which is essential for accurate clustering of data from heterogeneous tissues. We demonstrate the advantages of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Scalable supervised clustering methods such as RCA2 will facilitate unified analysis of cohort-scale SC datasets.
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Algoritmos , Análisis por Conglomerados , ARN Citoplasmático Pequeño/genética , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Animales , Artritis Reumatoide/genética , Células de la Médula Ósea/metabolismo , COVID-19/sangre , COVID-19/patología , Estudios de Cohortes , Conjuntos de Datos como Asunto , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Ratones , Especificidad de Órganos , Control de Calidad , RNA-Seq/normas , Análisis de la Célula Individual/normas , TranscriptomaRESUMEN
Single-cell and single-transcript measurement methods have elevated our ability to understand and engineer biological systems. However, defining and comparing performance between methods remains a challenge, in part due to the confounding effects of experimental variability. Here, we propose a generalizable framework for performing multiple methods in parallel using split samples, so that experimental variability is shared between methods. We demonstrate the utility of this framework by performing 12 different methods in parallel to measure the same underlying reference system for cellular response. We compare method performance using quantitative evaluations of bias and resolvability. We attribute differences in method performance to steps along the measurement process such as sample preparation, signal detection, and choice of measurand. Finally, we demonstrate how this framework can be used to benchmark different methods for single-transcript detection. The framework we present here provides a practical way to compare performance of any methods.
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Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Proteínas Bacterianas/genética , Sesgo , Bioingeniería , Escherichia coli/genética , Citometría de Flujo , Perfilación de la Expresión Génica/normas , Perfilación de la Expresión Génica/estadística & datos numéricos , Hibridación in Situ/métodos , Hibridación in Situ/normas , Hibridación in Situ/estadística & datos numéricos , Hibridación Fluorescente in Situ/métodos , Hibridación Fluorescente in Situ/normas , Hibridación Fluorescente in Situ/estadística & datos numéricos , Proteínas Luminiscentes/genética , Microscopía , ARN Bacteriano/análisis , Reproducibilidad de los Resultados , Análisis de la Célula Individual/normas , Análisis de la Célula Individual/estadística & datos numéricosRESUMEN
Genetic maps have been fundamental to building our understanding of disease genetics and evolutionary processes. The gametes of an individual contain all of the information required to perform a de novo chromosome-scale assembly of an individual's genome, which historically has been performed with populations and pedigrees. Here, we discuss how single-cell gamete sequencing offers the potential to merge the advantages of short-read sequencing with the ability to build personalized genetic maps and open up an entirely new space in personalized genetics.
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Genoma , Genómica/métodos , Células Germinativas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Medicina de Precisión/métodos , Análisis de la Célula Individual/métodos , Animales , Mapeo Cromosómico , Biología Computacional/métodos , Biología Computacional/normas , Interpretación Estadística de Datos , Heterogeneidad Genética , Genómica/normas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Medicina de Precisión/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de la Célula Individual/normas , Secuenciación Completa del GenomaRESUMEN
Normalization is an important step in the analysis of single-cell RNA-seq data. While no single method outperforms all others in all datasets, the choice of normalization can have profound impact on the results. Data-driven metrics can be used to rank normalization methods and select the best performers. Here, we show how to use R/Bioconductor to calculate normalization factors, apply them to compute normalized data, and compare several normalization approaches. Finally, we briefly show how to perform downstream analysis steps on the normalized data.
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RNA-Seq/normas , Análisis de la Célula Individual/normas , Animales , Biología Computacional/métodos , Biología Computacional/normas , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Control de Calidad , RNA-Seq/métodos , Estándares de Referencia , Análisis de Secuencia de ARN/métodos , Análisis de Secuencia de ARN/normas , Análisis de la Célula Individual/métodos , Programas Informáticos , Transcriptoma , Secuenciación del ExomaRESUMEN
Dimensionality reduction is a crucial step in essentially every single-cell RNA-sequencing (scRNA-seq) analysis. In this chapter, we describe the typical dimensionality reduction workflow that is used for scRNA-seq datasets, specifically highlighting the roles of principal component analysis, t-distributed stochastic neighborhood embedding, and uniform manifold approximation and projection in this setting. We particularly emphasize efficient computation; the software implementations used in this chapter can scale to datasets with millions of cells.