Single-cell atlases often include samples that span locations, laboratories and conditions, leading to complex, nested batch effects in data. Thus, joint analysis of atlas datasets requires reliable data integration. To guide integration method choice, we benchmarked 68 method and preprocessing combinations on 85 batches of gene expression, chromatin accessibility and simulation data from 23 publications, altogether representing >1.2 million cells distributed in 13 atlas-level integration tasks. We evaluated methods according to scalability, usability and their ability to remove batch effects while retaining biological variation using 14 evaluation metrics. We show that highly variable gene selection improves the performance of data integration methods, whereas scaling pushes methods to prioritize batch removal over conservation of biological variation. Overall, scANVI, Scanorama, scVI and scGen perform well, particularly on complex integration tasks, while single-cell ATAC-sequencing integration performance is strongly affected by choice of feature space. Our freely available Python module and benchmarking pipeline can identify optimal data integration methods for new data, benchmark new methods and improve method development.

Cell atlases often include samples that span locations, labs, and conditions, leading to complex, nested batch effects in data. Thus, joint analysis of atlas datasets requires reliable data integration .Choosing a data integration method is a challenge due to the difficulty of defining integration success. Here, we benchmark 38 method and preprocessing combinations on 77 batches of gene expression, chromatin accessibility, and simulation data from 23 publications, altogether representing >1.2 million cells distributed in nine atlas-level integration tasks. Our integration tasks span several common sources of variation such as individuals, species, and experimental labs. We evaluate methods according to scalability, usability, and their ability to remove batch effects while retaining biological variation.Using 14 evaluation metrics, we find that highly variable gene selection improves the performance of data integration methods, whereas scaling pushes methods to prioritize batch removal over conservation of biological variation. Overall, BBKNN, Scanorama, and scVI perform well, particularly on complex integration tasks; Seurat v3 performs well on simpler tasks with distinct biological signals; and methods that prioritize batch removal perform best for ATAC-seq data integration. Our freely available reproducible python module can be used to identify optimal data integration methods for new data, benchmark new methods, and improve method development.2

Organ- and body-scale cell atlases have the potential to transform our understanding of human biology. To capture the variability present in the population, these atlases must include diverse demographics such as age and ethnicity from both healthy and diseased individuals. The growth in both size and number of single-cell datasets, combined with recent advances in computational techniques, for the first time makes it possible to generate such comprehensive large-scale atlases through integration of multiple datasets. Here, we present the integrated Human Lung Cell Atlas (HLCA) combining 46 datasets of the human respiratory system into a single atlas spanning over 2.2 million cells from 444 individuals across health and disease. The HLCA contains a consensus re-annotation of published and newly generated datasets, resolving under- or misannotation of 59% of cells in the original datasets. The HLCA enables recovery of rare cell types, provides consensus marker genes for each cell type, and uncovers gene modules associated with demographic covariates and anatomical location within the respiratory system. To facilitate the use of the HLCA as a reference for single-cell lung research and allow rapid analysis of new data, we provide an interactive web portal to project datasets onto the HLCA. Finally, we demonstrate the value of the HLCA reference for interpreting disease-associated changes. Thus, the HLCA outlines a roadmap for the development and use of organ-scale cell atlases within the Human Cell Atlas.

Recent advances in single-cell technologies have enabled high-throughput molecular profiling of cells across modalities and locations. Single-cell transcriptomics data can now be complemented by chromatin accessibility, surface protein expression, adaptive immune receptor repertoire profiling and spatial information. The increasing availability of single-cell data across modalities has motivated the development of novel computational methods to help analysts derive biological insights. As the field grows, it becomes increasingly difficult to navigate the vast landscape of tools and analysis steps. Here, we summarize independent benchmarking studies of unimodal and multimodal single-cell analysis across modalities to suggest comprehensive best-practice workflows for the most common analysis steps. Where independent benchmarks are not available, we review and contrast popular methods. Our article serves as an entry point for novices in the field of single-cell (multi-)omic analysis and guides advanced users to the most recent best practices.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

Single-cell genomics is now producing an ever-increasing amount of datasets that, when integrated, could provide large-scale reference atlases of tissue in health as well as disease. Such large-scale atlases not only increase the scale and generalizability of analyses but also enable combining the knowledge generated by individual studies. Specifically, individual studies often differ in terms of cell annotation terminology and depth, with different groups specializing in different cell type compartments, often using distinct terminology. Understanding how these distinct sets of annotations are related and complement each other would mark a major step towards a consensus-based cell type annotation that reflects the latest knowledge in the field. Whereas recent computational techniques, referred to as 'reference mapping' methods, facilitate the usage and expansion of existing reference atlases by mapping new datasets (i.e. queries) onto an atlas; a systematic approach towards harmonizing dataset-specific cell type terminology and annotation depth is still lacking. Here, we present 'treeArches', a framework to automatically build and extend reference atlases while enriching them with an updatable hierarchy of cell type annotations across different datasets. We demonstrate various use cases for treeArches, from automatically resolving relations between reference and query cell types to identifying unseen cell types not present in the reference, such as disease-associated cell states. We envision treeArches enabling data-driven construction of consensus, atlas-level cell type hierarchies, as well as facilitating efficient usage of reference atlases.

Single-cell genomics is now producing an ever-increasing amount of datasets that, when integrated, could provide large-scale reference atlases of tissue in health and disease. Such large-scale atlases increase the scale and generalizability of analyses and enable combining knowledge generated by individual studies. Specifically, individual studies often differ regarding cell annotation terminology and depth, with different groups specializing in different cell type compartments, often using distinct terminology. Understanding how these distinct sets of annotations are related and complement each other would mark a major step towards a consensus-based cell-type annotation reflecting the latest knowledge in the field. Whereas recent computational techniques, referred to as ‘reference mapping’ methods, facilitate the usage and expansion of existing reference atlases by mapping new datasets (i.e. queries) onto an atlas; a systematic approach towards harmonizing dataset-specific cell-type terminology and annotation depth is still lacking. Here, we present ‘treeArches’, a framework to automatically build and extend reference atlases while enriching them with an updatable hierarchy of cell-type annotations across different datasets. We demonstrate various use cases for treeArches, from automatically resolving relations between reference and query cell types to identifying unseen cell types absent in the reference, such as disease-associated cell states. We envision treeArches enabling data-driven construction of consensus atlas-level cell-type hierarchies and facilitating efficient usage of reference atlases.

Immune checkpoint inhibitors (ICIs) enhance anticancer immunity by releasing repressive signals into tumor microenvironments (TMEs). To be effective, ICIs require preexisting immunologically “hot” niches for tumor antigen presentation and lymphocyte recruitment. How the mutational landscape of cancer cells shapes these immunological niches remains poorly defined. We found in human and murine colorectal cancer (CRC) models that the superior antitumor immune response of mismatch repair (MMR)–deficient CRC required tumor cell–intrinsic activation of cGAS-STING signaling triggered by genomic instability. Subsequently, we synthetically enforced STING signaling in CRC cells with intact MMR signaling using constitutively active STING variants. Even in MMR-proficient CRC, genetically encoded gain-of-function STING was sufficient to induce cancer cell–intrinsic interferon signaling, local activation of antigen-presenting cells, recruitment of effector lymphocytes, and sensitization of previously “cold” TMEs to ICI therapy in vivo. Thus, our results introduce a rational strategy for modulating cancer cell–intrinsic programs via engineered STING enforcement to sensitize resistant tumors to ICI responsiveness.