BACKGROUND: Alternative splicing contributes to molecular diversity across brain cell types. RNA-binding proteins (RBPs) regulate splicing, but the genome-wide mechanisms underlying cell-type-specific splicing remain poorly understood. RESULTS: Here, we want to unravel cell-type-specific splicing mechanisms by using RBP binding sites and/or the genomic sequence to predict exon inclusion in neurons and glia as measured by long-read single-cell data in the human hippocampus and frontal cortex. We found that exon inclusion of variable exons is harder to predict in neurons compared to glia in both brain regions. Comparing neurons and glia, the position of RBP binding sites in alternatively spliced exons in neurons differ more from non-variable exons indicating distinct splicing mechanisms. Model interpretation pinpointed RBPs, including QKI, potentially regulating alternative splicing between neurons and glia. Finally, we accurately predict and prioritize the effect of splicing QTLs. CONCLUSIONS: Our results indicate that the splicing mechanisms in variable exons in neurons diverged more from the standard mechanisms. Splicing in neurons might be less sequence-dependent and influenced more by, for instance, chromatin accessibility or methylation. Taken together, these results highlight new insights into the mechanisms regulating cell-type-specific alternative splicing in the brain.

The identification of expression quantitative trait loci (eQTLs) holds great potential to improve the interpretation of disease-associated genetic variation. As many such disease-associated variants act in a context-, tissue- or even cell-type-specific manner, single-cell RNA-sequencing (scRNA-seq) data is uniquely suitable for identifying the specific cell type or context in which these genetic variants act. However, due to the limited sample sizes in single-cell studies, discovery of cell-type-specific eQTLs is now limited. To improve power to detect such eQTLs, large-scale joint analyses are needed. These are however, complicated by privacy constraints due to sharing of genotype data and the measurement and technical variety across different scRNA-seq datasets as a result of differences in mRNA capture efficiency, experimental protocols, and sequencing strategies. A solution to these issues is a federated weighted meta-analysis (WMA) approach in which summary statistics are integrated using dataset-specific weights. Here, we compare different strategies and provide best practice recommendations for eQTL WMA across scRNA-seq datasets.

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.

Motivation: Knowing the relation between cell types is crucial for translating experimental results from mice to humans. Establishing cell type matches, however, is hindered by the biological differences between the species. A substantial amount of evolutionary information between genes that could be used to align the species is discarded by most of the current methods since they only use one-to-one orthologous genes. Some methods try to retain the information by explicitly including the relation between genes, however, not without caveats. Results: In this work, we present a model to transfer and align cell types in cross-species analysis (TACTiCS). First, TACTiCS uses a natural language processing model to match genes using their protein sequences. Next, TACTiCS employs a neural network to classify cell types within a species. Afterward, TACTiCS uses transfer learning to propagate cell type labels between species. We applied TACTiCS on scRNA-seq data of the primary motor cortex of human, mouse, and marmoset. Our model can accurately match and align cell types on these datasets. Moreover, our model outperforms Seurat and the state-of-the-art method SAMap. Finally, we show that our gene matching method results in better cell type matches than BLAST in our model.

Supervised methods are increasingly used to identify cell populations in single-cell data. Yet, current methods are limited in their ability to learn from multiple datasets simultaneously, are hampered by the annotation of datasets at different resolutions, and do not preserve annotations when retrained on new datasets. The latter point is especially important as researchers cannot rely on downstream analysis performed using earlier versions of the dataset. Here, we present scHPL, a hierarchical progressive learning method which allows continuous learning from single-cell data by leveraging the different resolutions of annotations across multiple datasets to learn and continuously update a classification tree. We evaluate the classification and tree learning performance using simulated as well as real datasets and show that scHPL can successfully learn known cellular hierarchies from multiple datasets while preserving the original annotations. scHPL is available at https://github.com/lcmmichielsen/scHPL.

Background: Single-cell transcriptomics is rapidly advancing our understanding of the cellular composition of complex tissues and organisms. A major limitation in most analysis pipelines is the reliance on manual annotations to determine cell identities, which are time-consuming and irreproducible. The exponential growth in the number of cells and samples has prompted the adaptation and development of supervised classification methods for automatic cell identification. Results: Here, we benchmarked 22 classification methods that automatically assign cell identities including single-cell-specific and general-purpose classifiers. The performance of the methods is evaluated using 27 publicly available single-cell RNA sequencing datasets of different sizes, technologies, species, and levels of complexity. We use 2 experimental setups to evaluate the performance of each method for within dataset predictions (intra-dataset) and across datasets (inter-dataset) based on accuracy, percentage of unclassified cells, and computation time. We further evaluate the methods' sensitivity to the input features, number of cells per population, and their performance across different annotation levels and datasets. We find that most classifiers perform well on a variety of datasets with decreased accuracy for complex datasets with overlapping classes or deep annotations. The general-purpose support vector machine classifier has overall the best performance across the different experiments. Conclusions: We present a comprehensive evaluation of automatic cell identification methods for single-cell RNA sequencing data. All the code used for the evaluation is available on GitHub (https://github.com/tabdelaal/scRNAseq-Benchmark). Additionally, we provide a Snakemake workflow to facilitate the benchmarking and to support the extension of new methods and new datasets.