Integration Of Single-cell Data Research Articles

DeepMap: Deep learning-based single-cell data integration using iterative cell matching and structure preservation constraints

DeepMap: Deep learning-based single-cell data integration using iterative cell matching and structure preservation constraints

Recovery of biological signals lost in single-cell batch integration with CellANOVA.

Data integration to align cells across batches has become a cornerstone of single-cell data analysis, critically affecting downstream results. Currently, there are no guidelines for when the biological differences between samples are separable from batch effects. Here we show that current paradigms for single-cell data integration remove biologically meaningful variation and introduce distortion. We present a statistical model and computationally scalable algorithm, CellANOVA (cell state space analysis of variance), that harnesses experimental design to explicitly recover biological signals that are erased during single-cell data integration. CellANOVA uses a 'pool-of-controls' design concept, applicable across diverse settings, to separate unwanted variation from biological variation of interest and allow the recovery of subtle biological signals. We apply CellANOVA to diverse contexts and validate the recovered biological signals by orthogonal assays. In particular, we show that CellANOVA is effective in the challenging case of single-cell and single-nucleus data integration, where it recovers subtle biological signals that can be validated and replicated by external data.

A Quantitative Computational Framework for Allopolyploid Single-Cell Data Integration and Core Gene Ranking in Development.

Polyploidization drives regulatory and phenotypic innovation. How the merger of different genomes contributes to polyploid development is a fundamental issue in evolutionary developmental biology and breeding research. Clarifying this issue is challenging because of genome complexity and the difficulty in tracking stochastic subgenome divergence during development. Recent single-cell sequencing techniques enabled probing subgenome-divergent regulation in the context of cellular differentiation. However, analyzing single-cell data suffers from high error rates due to high dimensionality, noise, and sparsity, and the errors stack up in polyploid analysis due to the increased dimensionality of comparisons between subgenomes of each cell, hindering deeper mechanistic understandings. In this study, we develop a quantitative computational framework, called "pseudo-genome divergence quantification" (pgDQ), for quantifying and tracking subgenome divergence directly at the cellular level. Further comparing with cellular differentiation trajectories derived from single-cell RNA sequencing data allows for an examination of the relationship between subgenome divergence and the progression of development. pgDQ produces robust results and is insensitive to data dropout and noise, avoiding high error rates due to multiple comparisons of genes, cells, and subgenomes. A statistical diagnostic approach is proposed to identify genes that are central to subgenome divergence during development, which facilitates the integration of different data modalities, enabling the identification of factors and pathways that mediate subgenome-divergent activity during development. Case studies have demonstrated that applying pgDQ to single-cell and bulk tissue transcriptomic data promotes a systematic and deeper understanding of how dynamic subgenome divergence contributes to developmental trajectories in polyploid evolution.

PIPET: predicting relevant subpopulations in single-cell data using phenotypic information from bulk data.

Single-cell RNA sequencing has revealed cellular heterogeneity in complex tissues, notably benefiting research on diseases such as cancer. However, the integration of single-cell data from small samples with extensive clinical features in bulk data remains underexplored. In this study, we introduce PIPET, an algorithmic method for predicting relevant subpopulations in single-cell data based on multivariate phenotypic information from bulk data. PIPET generates feature vectors for each phenotype from differentially expressed genes in bulk data and then identifies relevant cellular subpopulations by assessing the similarity between single-cell data and these vectors. Subsequently, phenotype-related cell states can be analyzed based on these subpopulations. In simulated datasets, PIPET showed robust performance in predicting multiclassification cellular subpopulations. Application of PIPET to lung adenocarcinoma single-cell RNA sequencing data revealed cellular subpopulations with poor survival and associations with TP53 mutations. Similarly, in breast cancer single-cell data, PIPET identified cellular subpopulations associated with the PAM50 clinical subtypes and triple-negative breast cancer subtypes. Overall, PIPET effectively identified relevant cellular subpopulations in single-cell data, guided by phenotypic information from bulk data. This approach comprehensively delineates the molecular characteristics of each cellular subpopulation, offering insights into disease-related subpopulations and guiding personalized treatment strategies.

WCN24-1387 Integration of Single Cell Data from Human and RNA-Seq Analysis from Rat to Identify Surrogate Drugs Mimicking the Effects of Metabolic Bariatric Surgery

WCN24-1387 Integration of Single Cell Data from Human and RNA-Seq Analysis from Rat to Identify Surrogate Drugs Mimicking the Effects of Metabolic Bariatric Surgery

Characterizing the impacts of dataset imbalance on single-cell data integration.

Computational methods for integrating single-cell transcriptomic data from multiple samples and conditions do not generally account for imbalances in the cell types measured in different datasets. In this study, we examined how differences in the cell types present, the number of cells per cell type and the cell type proportions across samples affect downstream analyses after integration. The Iniquitate pipeline assesses the robustness of integration results after perturbing the degree of imbalance between datasets. Benchmarking of five state-of-the-art single-cell RNA sequencing integration techniques in 2,600 integration experiments indicates that sample imbalance has substantial impacts on downstream analyses and the biological interpretation of integration results. Imbalance perturbation led to statistically significant variation in unsupervised clustering, cell type classification, differential expression and marker gene annotation, query-to-reference mapping and trajectory inference. We quantified the impacts of imbalance through newly introduced properties-aggregate cell type support and minimum cell type center distance. To better characterize and mitigate impacts of imbalance, we introduce balanced clustering metrics and imbalanced integration guidelines for integration method users.

Imbalanced single-cell data integration leads to loss of biological information.

Imbalanced single-cell data integration leads to loss of biological information.

Principled and interpretable alignability testing and integration of single-cell data

Single-cell data integration can provide a comprehensive molecular view of cells, and many algorithms have been developed to remove unwanted technical or biological variations and integrate heterogeneous single-cell datasets. Despite their wide usage, existing methods suffer from several fundamental limitations. In particular, we lack a rigorous statistical test for whether two high-dimensional single-cell datasets are alignable (and therefore should even be aligned). Moreover, popular methods can substantially distort the data during alignment, making the aligned data and downstream analysis difficult to interpret. To overcome these limitations, we present a spectral manifold alignment and inference (SMAI) framework, which enables principled and interpretable alignability testing and structure-preserving integration of single-cell data with the same type of features. SMAI provides a statistical test to robustly assess the alignability between datasets to avoid misleading inference and is justified by high-dimensional statistical theory. On a diverse range of real and simulated benchmark datasets, it outperforms commonly used alignment methods. Moreover, we show that SMAI improves various downstream analyses such as identification of differentially expressed genes and imputation of single-cell spatial transcriptomics, providing further biological insights. SMAI's interpretability also enables quantification and a deeper understanding of the sources of technical confounders in single-cell data.

Semi-supervised integration of single-cell transcriptomics data

Batch effects in single-cell RNA-seq data pose a significant challenge for comparative analyses across samples, individuals, and conditions. Although batch effect correction methods are routinely applied, data integration often leads to overcorrection and can result in the loss of biological variability. In this work we present STACAS, a batch correction method for scRNA-seq that leverages prior knowledge on cell types to preserve biological variability upon integration. Through an open-source benchmark, we show that semi-supervised STACAS outperforms state-of-the-art unsupervised methods, as well as supervised methods such as scANVI and scGen. STACAS scales well to large datasets and is robust to incomplete and imprecise input cell type labels, which are commonly encountered in real-life integration tasks. We argue that the incorporation of prior cell type information should be a common practice in single-cell data integration, and we provide a flexible framework for semi-supervised batch effect correction.

ScCorrector: a robust method for integrating multi-study single-cell data.

The advent of single-cell sequencing technologies has revolutionized cell biology studies. However, integrative analyses of diverse single-cell data face serious challenges, including technological noise, sample heterogeneity, and different modalities and species. To address these problems, we propose scCorrector, a variational autoencoder-based model that can integrate single-cell data from different studies and map them into a common space. Specifically, we designed a Study Specific Adaptive Normalization for each study in decoder to implement these features. scCorrector substantially achieves competitive and robust performance compared with state-of-the-art methods and brings novel insights under various circumstances (e.g. various batches, multi-omics, cross-species, and development stages). In addition, the integration of single-cell data and spatial data makes it possible to transfer information between different studies, which greatly expand the narrow range of genes covered by MERFISH technology. In summary, scCorrector can efficiently integrate multi-study single-cell datasets, thereby providing broad opportunities to tackle challenges emerging from noisy resources.

ScICML: Information-Theoretic Co-Clustering-Based Multi-View Learning for the Integrative Analysis of Single-Cell Multi-Omics Data.

Modern high-throughput sequencing technologies have enabled us to profile multiple molecular modalities from the same single cell, providing unprecedented opportunities to assay cellular heterogeneity from multiple biological layers. However, the datasets generated from these technologies tend to have high level of noise and are highly sparse, bringing challenges to data analysis. In this paper, we develop a novel information-theoretic co-clustering-based multi-view learning (scICML) method for multi-omics single-cell data integration. scICML utilizes co-clusterings to aggregate similar features for each view of data and uncover the common clustering pattern for cells. In addition, scICML automatically matches the clusters of the linked features across different data types for considering the biological dependency structure across different types of genomic features. Our experiments on four real-world datasets demonstrate that scICML improves the overall clustering performance and provides biological insights into the data analysis of peripheral blood mononuclear cells.

Accurate integration of single-cell DNA and RNA for analyzing intratumor heterogeneity using MaCroDNA

Cancers develop and progress as mutations accumulate, and with the advent of single-cell DNA and RNA sequencing, researchers can observe these mutations and their transcriptomic effects and predict proteomic changes with remarkable temporal and spatial precision. However, to connect genomic mutations with their transcriptomic and proteomic consequences, cells with either only DNA data or only RNA data must be mapped to a common domain. For this purpose, we present MaCroDNA, a method that uses maximum weighted bipartite matching of per-gene read counts from single-cell DNA and RNA-seq data. Using ground truth information from colorectal cancer data, we demonstrate the advantage of MaCroDNA over existing methods in accuracy and speed. Exemplifying the utility of single-cell data integration in cancer research, we suggest, based on results derived using MaCroDNA, that genomic mutations of large effect size increasingly contribute to differential expression between cells as Barrett’s esophagus progresses to esophageal cancer, reaffirming the findings of the previous studies.

Population-level integration of single-cell datasets enables multi-scale analysis across samples

The increasing generation of population-level single-cell atlases has the potential to link sample metadata with cellular data. Constructing such references requires integration of heterogeneous cohorts with varying metadata. Here we present single-cell population level integration (scPoli), an open-world learner that incorporates generative models to learn sample and cell representations for data integration, label transfer and reference mapping. We applied scPoli on population-level atlases of lung and peripheral blood mononuclear cells, the latter consisting of 7.8 million cells across 2,375 samples. We demonstrate that scPoli can explain sample-level biological and technical variations using sample embeddings revealing genes associated with batch effects and biological effects. scPoli is further applicable to single-cell sequencing assay for transposase-accessible chromatin and cross-species datasets, offering insights into chromatin accessibility and comparative genomics. We envision scPoli becoming an important tool for population-level single-cell data integration facilitating atlas use but also interpretation by means of multi-scale analyses.

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects.

Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A toward 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multimodal single-cell data integration, enable accurate identification of neuronal subtypes and uncover context-specific regulatory effects on cell-type-specific genes by TET enzymes.

Stabilized mosaic single-cell data integration using unshared features.

Currently available single-cell omics technologies capture many unique features with different biological information content. Data integration aims to place cells, captured with different technologies, onto a common embedding to facilitate downstream analytical tasks. Current horizontal data integration techniques use a set of common features, thereby ignoring non-overlapping features and losing information. Here we introduce StabMap, a mosaic data integration technique that stabilizes mapping of single-cell data by exploiting the non-overlapping features. StabMap first infers a mosaic data topology based on shared features, then projects all cells onto supervised or unsupervised reference coordinates by traversing shortest paths along the topology. We show that StabMap performs well in various simulation contexts, facilitates 'multi-hop' mosaic data integration where some datasets do not share any features and enables the use of spatial gene expression features for mapping dissociated single-cell data onto a spatial transcriptomic reference.

MASI enables fast model-free standardization and integration of single-cell transcriptomics data

Single-cell transcriptomics datasets from the same anatomical sites generated by different research labs are becoming increasingly common. However, fast and computationally inexpensive tools for standardization of cell-type annotation and data integration are still needed in order to increase research inclusivity. To standardize cell-type annotation and integrate single-cell transcriptomics datasets, we have built a fast model-free integration method, named MASI (Marker-Assisted Standardization and Integration). We benchmark MASI with other well-established methods and demonstrate that MASI outperforms other methods, in terms of integration, annotation, and speed. To harness knowledge from single-cell atlases, we demonstrate three case studies that cover integration across biological conditions, surveyed participants, and research groups, respectively. Finally, we show MASI can annotate approximately one million cells on a personal laptop, making large-scale single-cell data integration more accessible. We envision that MASI can serve as a cheap computational alternative for the single-cell research community.

GLUE multimodal single cell data.

A commentary on "Multi-omics single-cell data integration and regulatory inference with graph-linked embedding".

Cartography of Genomic Interactions Enables Deep Analysis of Single-Cell Expression Data

Remarkable advances in single cell genomics have presented unique challenges and opportunities for interrogating a wealth of biomedical inquiries. High dimensional genomic data are inherently complex because of intertwined relationships among the genes. Existing methods, including emerging deep learning-based approaches, do not consider the underlying biological characteristics during data processing, which greatly compromises the performance of data analysis and hinders the maximal utilization of state-of-the-art genomic techniques. In this work, we develop an entropy-based cartography strategy to contrive the high dimensional gene expression data into a configured image format, referred to as genomap, with explicit integration of the genomic interactions. This unique cartography casts the gene-gene interactions into the spatial configuration of genomaps and enables us to extract the deep genomic interaction features and discover underlying discriminative patterns of the data. We show that, for a wide variety of applications (cell clustering and recognition, gene signature extraction, single cell data integration, cellular trajectory analysis, dimensionality reduction, and visualization), the proposed approach drastically improves the accuracies of data analyses as compared to the state-of-the-art techniques.

Single-cell transcriptomics for the assessment of cardiac disease.

Cardiovascular disease is the leading cause of death globally. An advanced understanding of cardiovascular disease mechanisms is required to improve therapeutic strategies and patient risk stratification. State-of-the-art, large-scale, single-cell and single-nucleus transcriptomics facilitate the exploration of the cardiac cellular landscape at an unprecedented level, beyond its descriptive features, and can further our understanding of the mechanisms of disease and guide functional studies. In this Review, we provide an overview of the technical challenges in the experimental design of single-cell and single-nucleus transcriptomics studies, as well as a discussion of the type of inferences that can be made from the data derived from these studies. Furthermore, we describe novel findings derived from transcriptomics studies for each major cardiac cell type in both health and disease, and from development to adulthood. This Review also provides a guide to interpreting the exhaustive list of newly identified cardiac cell types and states, and highlights the consensus and discordances in annotation, indicating an urgent need for standardization. We describe advanced applications such as integration of single-cell data with spatial transcriptomics to map genes and cells on tissue and define cellular microenvironments that regulate homeostasis and disease progression. Finally, we discuss current and future translational and clinical implications of novel transcriptomics approaches, and provide an outlook of how these technologies will change the way we diagnose and treat heart disease.

A unified computational framework for single-cell data integration with optimal transport

Single-cell data integration can provide a comprehensive molecular view of cells. However, how to integrate heterogeneous single-cell multi-omics as well as spatially resolved transcriptomic data remains a major challenge. Here we introduce uniPort, a unified single-cell data integration framework that combines a coupled variational autoencoder (coupled-VAE) and minibatch unbalanced optimal transport (Minibatch-UOT). It leverages both highly variable common and dataset-specific genes for integration to handle the heterogeneity across datasets, and it is scalable to large-scale datasets. uniPort jointly embeds heterogeneous single-cell multi-omics datasets into a shared latent space. It can further construct a reference atlas for gene imputation across datasets. Meanwhile, uniPort provides a flexible label transfer framework to deconvolute heterogeneous spatial transcriptomic data using an optimal transport plan, instead of embedding latent space. We demonstrate the capability of uniPort by applying it to integrate a variety of datasets, including single-cell transcriptomics, chromatin accessibility, and spatially resolved transcriptomic data.