Highlights d Seurat v3 identifies correspondences between cells in different experiments d These ''anchors'' can be used to harmonize datasets into a single reference d Reference labels and data can be projected onto query datasets d Extends beyond RNA-seq to single-cell protein, chromatin, and spatial data

Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple data sets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq data sets based on common sources of variation, enabling the identification of shared populations across data sets and downstream comparative analysis. We apply this approach, implemented in our R toolkit Seurat (http://satijalab.org/seurat/), to align scRNA-seq data sets of peripheral blood mononuclear cells under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across data sets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq data sets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution.

Summary The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce “weighted-nearest neighbor” analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Single cell transcriptomics (scRNA-seq) has transformed our ability to discover and annotate cell types and states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, including high-dimensional immunophenotypes, chromatin accessibility, and spatial positioning, a key analytical challenge is to integrate these datasets into a harmonized atlas that can be used to better understand cellular identity and function. Here, we develop a computational strategy to "anchor" diverse datasets together, enabling us to integrate and compare single cell measurements not only across scRNA-seq technologies, but different modalities as well. After demonstrating substantial improvement over existing methods for data integration, we anchor scRNA-seq experiments with scATAC-seq datasets to explore chromatin differences in closely related interneuron subsets, and project single cell protein measurements onto a human bone marrow atlas to annotate and characterize lymphocyte populations. Lastly, we demonstrate how anchoring can harmonize in-situ gene expression and scRNA-seq datasets, allowing for the transcriptome-wide imputation of spatial gene expression patterns, and the identification of spatial relationships between mapped cell types in the visual cortex. Our work presents a strategy for comprehensive integration of single cell data, including the assembly of harmonized references, and the transfer of information across datasets.Availability: Installation instructions, documentation, and tutorials are available at: https://www.satijalab.org/seurat effective, they can also struggle in cases where only a subset of cell types are shared across datasets, or significant technical variation masks shared biological signal. Additionally, these methods focus on scRNA-seq and are not designed to integrate information across different modalities, nor do they enable the transfer of information from one dataset to another.Here, we present a unified strategy for reference assembly and transfer learning for transcriptomic, epigenomic, proteomic, and spatially-resolved single cell data. Through the identification of cell pairwise correspondences between single cells across datasets, termed "anchors", we can transform datasets into a shared space, even in the presence of extensive technical and/or biological differences. This enables the construction of harmonized atlases at the tissue or organismal scale. These anchors also enable effective transfer of discrete or continuous data from a reference onto a query dataset. This allows for the transfer of cell labels learned from scRNA-seq onto scATAC-seq data to explore differences in the regulatory landscape between distinct interneuron subsets, and the transfer of protein measurements 3 onto massive public resources to characterize lymphoid populations in human bone marrow. Finally, the anchoring of STARmap and scRNA-seq datasets enables the transcriptome-wide imputation of spatial gene expression pattern...

Advances in single-cell RNA sequencing (scRNA-seq) have allowed for comprehensive analysis of the immune system. In this Review, we briefly describe the available scRNA-seq technologies together with their corresponding strengths and weaknesses. We discuss in depth how scRNA-seq can be used to deconvolve immune system heterogeneity by identifying novel distinct immune cell subsets in health and disease, characterizing stochastic heterogeneity within a cell population and building developmental 'trajectories' for immune cells. Finally, we discuss future directions of the field and present integrated approaches to complement molecular information from a single cell with studies of the environment, epigenetic state and cell lineage.

The simultaneous measurement of multiple modalities, known as multimodal analysis, represents an exciting frontier for single-cell genomics and necessitates new computational methods that can define cellular states based on multiple data types. Here, we introduce "weighted-nearest neighbor analysis", an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of hundreds of thousands of human white blood cells alongside a panel of 228 antibodies to construct a multimodal reference atlas of the circulating immune system. We demonstrate that integrative analysis substantially improves our ability to resolve cell states and validate the presence of previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets, and to interpret immune responses to vaccination and COVID-19. Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets, including paired measurements of RNA and chromatin state, and to look beyond the transcriptome towards a unified and multimodal definition of cellular identity. Availability: Installation instructions, documentation, tutorials, and CITE-seq datasets are available at http://www.satijalab.org/seurat

Multi-modal single-cell assays provide high-resolution snapshots of complex cell populations but are mostly limited to transcriptome plus an additional modality. Here, we describe Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct sgRNA capture and to clonotype-aware multimodal phenotyping of cancer samples.

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