RESUMO
Gene regulatory elements, such as enhancers, greatly influence cell identity by tuning the transcriptional activity of specific cell types. Dynamics of enhancer landscape during early human Th17 cell differentiation remains incompletely understood. Leveraging ATAC-seq-based profiling of chromatin accessibility and comprehensive analysis of key histone marks, we identified a repertoire of enhancers that potentially exert control over the fate specification of Th17 cells. We found 23 SNPs associated with autoimmune diseases within Th17-enhancers that precisely overlapped with the binding sites of transcription factors actively engaged in T-cell functions. Among the Th17-specific enhancers, we identified an enhancer in the intron of RORA and demonstrated that this enhancer positively regulates RORA transcription. Moreover, CRISPR-Cas9-mediated deletion of a transcription factor binding site-rich region within the identified RORA enhancer confirmed its role in regulating RORA transcription. These findings provide insights into the potential mechanism by which the RORA enhancer orchestrates Th17 differentiation.
Assuntos
Diferenciação Celular , Elementos Facilitadores Genéticos , Células Th17 , Humanos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Elementos Facilitadores Genéticos/genética , Células Th17/imunologia , Polimorfismo de Nucleotídeo Único , Regulação da Expressão Gênica , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Sítios de Ligação/genética , Sistemas CRISPR-CasRESUMO
Single-cell RNA-sequencing (scRNA-seq) enables researchers to quantify transcriptomes of thousands of cells simultaneously and study transcriptomic changes between cells. scRNA-seq datasets increasingly include multisubject, multicondition experiments to investigate cell-type-specific differential states (DS) between conditions. This can be performed by first identifying the cell types in all the subjects and then by performing a DS analysis between the conditions within each cell type. Naïve single-cell DS analysis methods that treat cells statistically independent are subject to false positives in the presence of variation between biological replicates, an issue known as the pseudoreplicate bias. While several methods have already been introduced to carry out the statistical testing in multisubject scRNA-seq analysis, comparisons that include all these methods are currently lacking. Here, we performed a comprehensive comparison of 18 methods for the identification of DS changes between conditions from multisubject scRNA-seq data. Our results suggest that the pseudobulk methods performed generally best. Both pseudobulks and mixed models that model the subjects as a random effect were superior compared with the naïve single-cell methods that do not model the subjects in any way. While the naïve models achieved higher sensitivity than the pseudobulk methods and the mixed models, they were subject to a high number of false positives. In addition, accounting for subjects through latent variable modeling did not improve the performance of the naïve methods.
Assuntos
Benchmarking , Perfilação da Expressão Gênica , Perfilação da Expressão Gênica/métodos , Humanos , RNA , RNA-Seq , Análise de Sequência de RNA/métodosRESUMO
MOTIVATION: Single-cell RNA-sequencing enables cell-level investigation of cell differentiation, which can be modelled using trajectory inference methods. While tremendous effort has been put into designing these methods, inferring accurate trajectories automatically remains difficult. Therefore, the standard approach involves testing different trajectory inference methods and picking the trajectory giving the most biologically sensible model. As the default parameters are often suboptimal, their tuning requires methodological expertise. RESULTS: We introduce Totem, an open-source, easy-to-use R package designed to facilitate inference of tree-shaped trajectories from single-cell data. Totem generates a large number of clustering results, estimates their topologies as minimum spanning trees, and uses them to measure the connectivity of the cells. Besides automatic selection of an appropriate trajectory, cell connectivity enables to visually pinpoint branching points and milestones relevant to the trajectory. Furthermore, testing different trajectories with Totem is fast, easy, and does not require in-depth methodological knowledge. AVAILABILITY AND IMPLEMENTATION: Totem is available as an R package at https://github.com/elolab/Totem.
Assuntos
Diferenciação Celular , Análise por ConglomeradosRESUMO
MOTIVATION: Computational models are needed to infer a representation of the cells, i.e. a trajectory, from single-cell RNA-sequencing data that model cell differentiation during a dynamic process. Although many trajectory inference methods exist, their performance varies greatly depending on the dataset and hence there is a need to establish more accurate, better generalizable methods. RESULTS: We introduce scShaper, a new trajectory inference method that enables accurate linear trajectory inference. The ensemble approach of scShaper generates a continuous smooth pseudotime based on a set of discrete pseudotimes. We demonstrate that scShaper is able to infer accurate trajectories for a variety of trigonometric trajectories, including many for which the commonly used principal curves method fails. A comprehensive benchmarking with state-of-the-art methods revealed that scShaper achieved superior accuracy of the cell ordering and, in particular, the differentially expressed genes. Moreover, scShaper is a fast method with few hyperparameters, making it a promising alternative to the principal curves method for linear pseudotemporal ordering. AVAILABILITY AND IMPLEMENTATION: scShaper is available as an R package at https://github.com/elolab/scshaper. The test data are available at https://doi.org/10.5281/zenodo.5734488. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Análise da Expressão Gênica de Célula Única , Software , Análise de Célula Única/métodos , Diferenciação Celular/genética , Sequenciamento do Exoma , Análise de Sequência de RNA/métodosRESUMO
MOTIVATION: Single-cell RNA-seq allows researchers to identify cell populations based on unsupervised clustering of the transcriptome. However, subpopulations can have only subtle transcriptomic differences and the high dimensionality of the data makes their identification challenging. RESULTS: We introduce ILoReg, an R package implementing a new cell population identification method that improves identification of cell populations with subtle differences through a probabilistic feature extraction step that is applied before clustering and visualization. The feature extraction is performed using a novel machine learning algorithm, called iterative clustering projection (ICP), that uses logistic regression and clustering similarity comparison to iteratively cluster data. Remarkably, ICP also manages to integrate feature selection with the clustering through L1-regularization, enabling the identification of genes that are differentially expressed between cell populations. By combining solutions of multiple ICP runs into a single consensus solution, ILoReg creates a representation that enables investigating cell populations with a high resolution. In particular, we show that the visualization of ILoReg allows segregation of immune and pancreatic cell populations in a more pronounced manner compared with current state-of-the-art methods. AVAILABILITY AND IMPLEMENTATION: ILoReg is available as an R package at https://bioconductor.org/packages/ILoReg. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Transcriptoma , Análise por Conglomerados , Perfilação da Expressão Gênica , RNA-Seq , Análise de Sequência de RNA , Análise de Célula Única , Software , Sequenciamento do ExomaRESUMO
BACKGROUND: Detection of copy number variations (CNVs) from high-throughput next-generation whole-genome sequencing (WGS) data has become a widely used research method during the recent years. However, only a little is known about the applicability of the developed algorithms to ultra-low-coverage (0.0005-0.8×) data that is used in various research and clinical applications, such as digital karyotyping and single-cell CNV detection. RESULT: Here, the performance of six popular read-depth based CNV detection algorithms (BIC-seq2, Canvas, CNVnator, FREEC, HMMcopy, and QDNAseq) was studied using ultra-low-coverage WGS data. Real-world array- and karyotyping kit-based validation were used as a benchmark in the evaluation. Additionally, ultra-low-coverage WGS data was simulated to investigate the ability of the algorithms to identify CNVs in the sex chromosomes and the theoretical minimum coverage at which these tools can accurately function. Our results suggest that while all the methods were able to detect large CNVs, many methods were susceptible to producing false positives when smaller CNVs (< 2 Mbp) were detected. There was also significant variability in their ability to identify CNVs in the sex chromosomes. Overall, BIC-seq2 was found to be the best method in terms of statistical performance. However, its significant drawback was by far the slowest runtime among the methods (> 3 h) compared with FREEC (~ 3 min), which we considered the second-best method. CONCLUSIONS: Our comparative analysis demonstrates that CNV detection from ultra-low-coverage WGS data can be a highly accurate method for the detection of large copy number variations when their length is in millions of base pairs. These findings facilitate applications that utilize ultra-low-coverage CNV detection.
Assuntos
Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Algoritmos , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Deciphering the meaning of the human DNA is an outstanding goal which would revolutionize medicine and our way for treating diseases. In recent years, non-coding RNAs have attracted much attention and shown to be functional in part. Yet the importance of these RNAs especially for higher biological functions remains under investigation. METHODS: In this paper, we analyze RNA-seq data, including non-coding and protein coding RNAs, from lung adenocarcinoma patients, a histologic subtype of non-small-cell lung cancer, with deep learning neural networks and other state-of-the-art classification methods. The purpose of our paper is three-fold. First, we compare the classification performance of different versions of deep belief networks with SVMs, decision trees and random forests. Second, we compare the classification capabilities of protein coding and non-coding RNAs. Third, we study the influence of feature selection on the classification performance. RESULTS: As a result, we find that deep belief networks perform at least competitively to other state-of-the-art classifiers. Second, data from non-coding RNAs perform better than coding RNAs across a number of different classification methods. This demonstrates the equivalence of predictive information as captured by non-coding RNAs compared to protein coding RNAs, conventionally used in computational diagnostics tasks. Third, we find that feature selection has in general a negative effect on the classification performance which means that unfiltered data with all features give the best classification results. CONCLUSIONS: Our study is the first to use ncRNAs beyond miRNAs for the computational classification of cancer and for performing a direct comparison of the classification capabilities of protein coding RNAs and non-coding RNAs.
Assuntos
Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , Biologia Computacional/métodos , Árvores de Decisões , Humanos , Neoplasias Pulmonares/patologia , Aprendizado de Máquina , MicroRNAs/genética , Redes Neurais de Computação , RNA Mensageiro/genética , Análise de Sequência de RNA/métodosRESUMO
Single-cell transcriptomics allows unbiased characterization of cell heterogeneity in a sample by profiling gene expression at single-cell level. These profiles capture snapshots of transient or steady states in dynamic processes, such as cell cycle, activation, or differentiation, which can be computationally ordered into a "flip-book" of cell development using trajectory inference methods. However, prediction of more complex topology structures, such as multifurcations or trees, remains challenging. In this chapter, we present two user-friendly protocols for inferring tree-shaped single-cell trajectories and pseudotime from single-cell transcriptomics data with Totem. Totem is a trajectory inference method that offers flexibility in inferring both nonlinear and linear trajectories and usability by avoiding the cumbersome fine-tuning of parameters. The QuickStart protocol provides a simple and practical example, whereas the GuidedStart protocol details the analysis step-by-step. Both protocols are demonstrated using a case study of human bone marrow CD34+ cells, allowing the study of the branching of three lineages: erythroid, lymphoid, and myeloid. All the analyses can be fully reproduced in Linux, macOS, and Windows operating systems (amd64 architecture) with >8 Gb of RAM using the provided docker image distributed with notebooks, scripts, and data in Docker Hub (elolab/repro-totem-ti). These materials are shared online under open-source license at https://elolab.github.io/Totem-protocol .
Assuntos
Análise de Célula Única , Software , Análise de Célula Única/métodos , Humanos , Perfilação da Expressão Gênica/métodos , Biologia Computacional/métodos , Transcriptoma , Linhagem da Célula/genética , Algoritmos , Diferenciação CelularRESUMO
Somatic mutations in T cells can cause cancer but also have implications for immunological diseases and cell therapies. The mutation spectrum in nonmalignant T cells is unclear. Here, we examined somatic mutations in CD4+ and CD8+ T cells from 90 patients with hematological and immunological disorders and used T cell receptor (TCR) and single-cell sequencing to link mutations with T cell expansions and phenotypes. CD8+ cells had a higher mutation burden than CD4+ cells. Notably, the biggest variant allele frequency (VAF) of non-synonymous variants was higher than synonymous variants in CD8+ T cells, indicating non-random occurrence. The non-synonymous VAF in CD8+ T cells strongly correlated with the TCR frequency, but not age. We identified mutations in pathways essential for T cell function and often affected lymphoid neoplasia. Single-cell sequencing revealed cytotoxic TEMRA phenotypes of mutated T cells. Our findings suggest that somatic mutations contribute to CD8+ T cell expansions without malignant transformation.
Assuntos
Linfócitos T CD8-Positivos , Mutação , Receptores de Antígenos de Linfócitos T , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Adulto , Análise de Célula Única , Masculino , Feminino , Pessoa de Meia-Idade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Frequência do Gene , Fenótipo , IdosoRESUMO
The serine/threonine-specific Moloney murine leukemia virus (PIM) kinase family (i.e., PIM1, PIM2, and PIM3) has been extensively studied in tumorigenesis. PIM kinases are downstream of several cytokine signaling pathways that drive immune-mediated diseases. Uncontrolled T helper 17 (Th17) cell activation has been associated with the pathogenesis of autoimmunity. However, the detailed molecular function of PIMs in human Th17 cell regulation has yet to be studied. In the present study, we comprehensively investigated how the three PIMs simultaneously alter transcriptional gene regulation during early human Th17 cell differentiation. By combining PIM triple knockdown with bulk and scRNA-seq approaches, we found that PIM deficiency promotes the early expression of key Th17-related genes while suppressing Th1-lineage genes. Further, PIMs modulate Th cell signaling, potentially via STAT1 and STAT3. Overall, our study highlights the inhibitory role of PIMs in human Th17 cell differentiation, thereby suggesting their association with autoimmune phenotypes.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Animais , Camundongos , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Transdução de Sinais , Hematopoese , Diferenciação Celular , Células Th17/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fimmu.2020.578848.].
RESUMO
Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRß) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRß signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients' repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients.
Assuntos
Anticorpos Antiproteína Citrulinada/sangue , Artrite Reumatoide/genética , Citocinas/genética , Genes Codificadores dos Receptores de Linfócitos T , Linfócitos T Citotóxicos/metabolismo , Transcriptoma , Adolescente , Adulto , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , RNA-Seq , Índice de Gravidade de Doença , Análise de Célula Única , Linfócitos T Citotóxicos/imunologia , Sequenciamento do Exoma , Adulto JovemRESUMO
Genomics data provide great opportunities for translational research and the clinical practice, for example, for predicting disease stages. However, the classification of such data is a challenging task due to their high dimensionality, noise, and heterogeneity. In recent years, deep learning classifiers generated much interest, but due to their complexity, so far, little is known about the utility of this method for genomics. In this paper, we address this problem by studying a computational diagnostics task by classification of breast cancer and inflammatory bowel disease patients based on high-dimensional gene expression data. We provide a comprehensive analysis of the classification performance of deep belief networks (DBNs) in dependence on its multiple model parameters and in comparison with support vector machines (SVMs). Furthermore, we investigate combined classifiers that integrate DBNs with SVMs. Such a classifier utilizes a DBN as representation learner forming the input for a SVM. Overall, our results provide guidelines for the complex usage of DBN for classifying gene expression data from complex diseases.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de DNA/métodos , Inteligência Artificial , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Biologia Computacional/métodos , Aprendizado Profundo , Feminino , Expressão Gênica , Genômica , Humanos , Doenças Inflamatórias Intestinais/classificação , Doenças Inflamatórias Intestinais/genética , Aprendizado de Máquina , Masculino , Redes Neurais de Computação , Máquina de Vetores de SuporteRESUMO
BACKGROUND: Alzheimer's disease results from a neurodegenerative process that starts well before the diagnosis can be made. New prognostic or diagnostic markers enabling early intervention into the disease process would be highly valuable. Environmental and lifestyle factors largely modulate the disease risk and may influence the pathogenesis through epigenetic mechanisms, such as DNA methylation. As environmental and lifestyle factors may affect multiple tissues of the body, we hypothesized that the disease-associated DNA methylation signatures are detectable in the peripheral blood of discordant twin pairs. RESULTS: Comparison of 23 disease discordant Finnish twin pairs with reduced representation bisulfite sequencing revealed peripheral blood DNA methylation differences in 11 genomic regions with at least 15.0% median methylation difference and FDR adjusted p value ≤ 0.05. Several of the affected genes are primarily associated with neuronal functions and pathologies and do not display disease-associated differences in gene expression in blood. The DNA methylation mark in ADARB2 gene was found to be differentially methylated also in the anterior hippocampus, including entorhinal cortex, of non-twin cases and controls. Targeted bisulfite pyrosequencing of the DNA methylation mark in ADARB2 gene in 62 Finnish and Swedish twin pairs revealed that, in addition to the disease status, DNA methylation of this region is influenced by gender, age, zygosity, APOE genotype, and smoking. Further analysis of 120 Swedish twin pairs indicated that this specific DNA methylation mark is not predictive for Alzheimer's disease and becomes differentially methylated after disease onset. CONCLUSIONS: DNA methylation differences can be detected in the peripheral blood of twin pairs discordant for Alzheimer's disease. These DNA methylation signatures may have value as disease markers and provide insights into the molecular mechanisms of pathogenesis. We found no evidence that the DNA methylation marks would be associated with gene expression in blood. Further studies are needed to elucidate the potential importance of the associated genes in neuronal functions and to validate the prognostic or diagnostic value of the individual marks or marker panels.