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The rapid development of single-cell RNA sequencing (scRNA-seq) technology allows us to study gene expression heterogeneity at the cellular level. Cell annotation is the basis for subsequent downstream analysis in single-cell data mining. As more and more well-annotated scRNA-seq reference data become available, many automatic annotation methods have sprung up in order to simplify the cell annotation process on unlabeled target data. However, existing methods rarely explore the fine-grained semantic knowledge of novel cell types absent from the reference data, and they are usually susceptible to batch effects on the classification of seen cell types. Taking into consideration the limitations above, this paper proposes a new and practical task called generalized cell type annotation and discovery for scRNA-seq data whereby target cells are labeled with either seen cell types or cluster labels, instead of a unified 'unassigned' label. To accomplish this, we carefully design a comprehensive evaluation benchmark and propose a novel end-to-end algorithmic framework called scGAD. Specifically, scGAD first builds the intrinsic correspondences on seen and novel cell types by retrieving geometrically and semantically mutual nearest neighbors as anchor pairs. Together with the similarity affinity score, a soft anchor-based self-supervised learning module is then designed to transfer the known label information from reference data to target data and aggregate the new semantic knowledge within target data in the prediction space. To enhance the inter-type separation and intra-type compactness, we further propose a confidential prototype self-supervised learning paradigm to implicitly capture the global topological structure of cells in the embedding space. Such a bidirectional dual alignment mechanism between embedding space and prediction space can better handle batch effect and cell type shift. Extensive results on massive simulation datasets and real datasets demonstrate the superiority of scGAD over various state-of-the-art clustering and annotation methods. We also implement marker gene identification to validate the effectiveness of scGAD in clustering novel cell types and their biological significance. To the best of our knowledge, we are the first to introduce this new and practical task and propose an end-to-end algorithmic framework to solve it. Our method scGAD is implemented in Python using the Pytorch machine-learning library, and it is freely available at https://github.com/aimeeyaoyao/scGAD. [ABSTRACT FROM AUTHOR]

Single-cell sequencing technologies are widely used to discover the evolutionary relationships and the differences in cells. Since dropout events may frustrate the analysis, many imputation approaches for single-cell RNA-seq data have appeared in previous attempts. However, previous imputation attempts usually suffer from the over-smooth problem, which may bring limited improvement or negative effect for the downstream analysis of single-cell RNA-seq data. To solve this difficulty, we propose a novel two-stage diffusion-denoising method called SCDD for large-scale single-cell RNA-seq imputation in this paper. We introduce the diffusion i.e. a direct imputation strategy using the expression of similar cells for potential dropout sites, to perform the initial imputation at first. After the diffusion, a joint model integrated with graph convolutional neural network and contractive autoencoder is developed to generate superposition states of similar cells, from which we restore the original states and remove the noise introduced by the diffusion. The final experimental results indicate that SCDD could effectively suppress the over-smooth problem and remarkably improve the effect of single-cell RNA-seq downstream analysis, including clustering and trajectory analysis. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing has enabled to capture the gene activities at single-cell resolution, thus allowing reconstruction of cell-type-specific gene regulatory networks (GRNs). The available algorithms for reconstructing GRNs are commonly designed for bulk RNA-seq data, and few of them are applicable to analyze scRNA-seq data by dealing with the dropout events and cellular heterogeneity. In this paper, we represent the joint gene expression distribution of a gene pair as an image and propose a novel supervised deep neural network called DeepDRIM which utilizes the image of the target TF-gene pair and the ones of the potential neighbors to reconstruct GRN from scRNA-seq data. Due to the consideration of TF-gene pair's neighborhood context, DeepDRIM can effectively eliminate the false positives caused by transitive gene–gene interactions. We compared DeepDRIM with nine GRN reconstruction algorithms designed for either bulk or single-cell RNA-seq data. It achieves evidently better performance for the scRNA-seq data collected from eight cell lines. The simulated data show that DeepDRIM is robust to the dropout rate, the cell number and the size of the training data. We further applied DeepDRIM to the scRNA-seq gene expression of B cells from the bronchoalveolar lavage fluid of the patients with mild and severe coronavirus disease 2019. We focused on the cell-type-specific GRN alteration and observed targets of TFs that were differentially expressed between the two statuses to be enriched in lysosome, apoptosis, response to decreased oxygen level and microtubule, which had been proved to be associated with coronavirus infection. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) has enabled us to study biological questions at the single-cell level. Currently, many analysis tools are available to better utilize these relatively noisy data. In this review, we summarize the most widely used methods for critical downstream analysis steps (i.e. clustering, trajectory inference, cell-type annotation and integrating datasets). The advantages and limitations are comprehensively discussed, and we provide suggestions for choosing proper methods in different situations. We hope this paper will be useful for scRNA-seq data analysts and bioinformatics tool developers. [ABSTRACT FROM AUTHOR]

Clustering analysis is widely used in single-cell ribonucleic acid (RNA)-sequencing (scRNA-seq) data to discover cell heterogeneity and cell states. While many clustering methods have been developed for scRNA-seq analysis, most of these methods require to provide the number of clusters. However, it is not easy to know the exact number of cell types in advance, and experienced determination is not always reliable. Here, we have developed ADClust, an automatic deep embedding clustering method for scRNA-seq data, which can accurately cluster cells without requiring a predefined number of clusters. Specifically, ADClust first obtains low-dimensional representation through pre-trained autoencoder and uses the representations to cluster cells into initial micro-clusters. The clusters are then compared in between by a statistical test, and similar micro-clusters are merged into larger clusters. According to the clustering, cell representations are updated so that each cell will be pulled toward centers of its assigned cluster and similar clusters, while cells are separated to keep distances between clusters. This is accomplished through jointly optimizing the carefully designed clustering and autoencoder loss functions. This merging process continues until convergence. ADClust was tested on 11 real scRNA-seq datasets and was shown to outperform existing methods in terms of both clustering performance and the accuracy on the number of the determined clusters. More importantly, our model provides high speed and scalability for large datasets. [ABSTRACT FROM AUTHOR]

Single-cell RNA-sequencing (scRNA-seq) is being used extensively to measure the mRNA expression of individual cells from deconstructed tissues, organs and even entire organisms to generate cell atlas references, leading to discoveries of novel cell types and deeper insight into biological trajectories. These massive datasets are usually collected from many samples using different scRNA-seq technology platforms, including the popular SMART-Seq2 (SS2) and 10X platforms. Inherent heterogeneities between platforms, tissues and other batch effects make scRNA-seq data difficult to compare and integrate, especially in large-scale cell atlas efforts; yet, accurate integration is essential for gaining deeper insights into cell biology. We present FIRM, a re-scaling algorithm which accounts for the effects of cell type compositions, and achieve accurate integration of scRNA-seq datasets across multiple tissue types, platforms and experimental batches. Compared with existing state-of-the-art integration methods, FIRM provides accurate mixing of shared cell type identities and superior preservation of original structure without overcorrection, generating robust integrated datasets for downstream exploration and analysis. FIRM is also a facile way to transfer cell type labels and annotations from one dataset to another, making it a reliable and versatile tool for scRNA-seq analysis, especially for cell atlas data integration. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) techniques provide high-resolution data on cellular heterogeneity in diverse tissues, and a critical step for the data analysis is cell type identification. Traditional methods usually cluster the cells and manually identify cell clusters through marker genes, which is time-consuming and subjective. With the launch of several large-scale single-cell projects, millions of sequenced cells have been annotated and it is promising to transfer labels from the annotated datasets to newly generated datasets. One powerful way for the transferring is to learn cell relations through the graph neural network (GNN), but traditional GNNs are difficult to process millions of cells due to the expensive costs of the message-passing procedure at each training epoch. Here, we have developed a robust and scalable GNN-based method for accurate single-cell classification (GraphCS), where the graph is constructed to connect similar cells within and between labelled and unlabeled scRNA-seq datasets for propagation of shared information. To overcome the slow information propagation of GNN at each training epoch, the diffused information is pre-calculated via the approximate Generalized PageRank algorithm, enabling sublinear complexity over cell numbers. Compared with existing methods, GraphCS demonstrates better performance on simulated, cross-platform, cross-species and cross-omics scRNA-seq datasets. More importantly, our model provides a high speed and scalability on large datasets, and can achieve superior performance for 1 million cells within 50 min. [ABSTRACT FROM AUTHOR]

Single-cell RNA sequencing (scRNA-seq) has enabled researchers to study gene expression at the cellular level. However, due to the extremely low levels of transcripts in a single cell and technical losses during reverse transcription, gene expression at a single-cell resolution is usually noisy and highly dimensional; thus, statistical analyses of single-cell data are a challenge. Although many scRNA-seq data analysis tools are currently available, a gold standard pipeline is not available for all datasets. Therefore, a general understanding of bioinformatics and associated computational issues would facilitate the selection of appropriate tools for a given set of data. In this review, we provide an overview of the goals and most popular computational analysis tools for the quality control, normalization, imputation, feature selection and dimension reduction of scRNA-seq data. [ABSTRACT FROM AUTHOR]

Accumulating studies demonstrated that the roles of lncRNAs for tumorigenesis were isoform-dependent and their aberrant splicing patterns in cancers contributed to function specificity. However, there is no existing database focusing on cancer-related alternative splicing of lncRNAs. Here, we developed a comprehensive database called LncAS2Cancer, which collected 5335 bulk RNA sequencing and 1826 single-cell RNA sequencing samples, covering over 30 cancer types. By applying six state-of-the-art splicing algorithms, 50 859 alternative splicing events for 8 splicing types were identified and deposited in the database. In addition, the database contained the following information: (i) splicing patterns of lncRNAs under seven different conditions, such as gene interference, which facilitated to infer potential regulators; (ii) annotation information derived from eight sources and manual curation, to understand the functional impact of affected sequences; (iii) survival analysis to explore potential biomarkers; as well as (iv) a suite of tools to browse, search, visualize and download interesting information. LncAS2Cancer could not only confirm the known cancer-associated lncRNA isoforms but also indicate novel ones. Using the data deposited in LncAS2Cancer, we compared gene model and transcript overlap between lncRNAs and protein-coding genes and discusses how these factors, along with sequencing depth, affected the interpretation of splicing signals. Based on recurrent signals and potential confounders, we proposed a reliable score to prioritize splicing events for further elucidation. Together, with the broad collection of lncRNA splicing patterns and annotation, LncAS2Cancer will provide important new insights into the diverse functional roles of lncRNA isoforms in human cancers. LncAS2Cancer is freely available at https://lncrna2as.cd120.com/. [ABSTRACT FROM AUTHOR]

Batch effect correction has been recognized to be indispensable when integrating single-cell RNA sequencing (scRNA-seq) data from multiple batches. State-of-the-art methods ignore single-cell cluster label information, but such information can improve the effectiveness of batch effect correction, particularly under realistic scenarios where biological differences are not orthogonal to batch effects. To address this issue, we propose SMNN for batch effect correction of scRNA-seq data via supervised mutual nearest neighbor detection. Our extensive evaluations in simulated and real datasets show that SMNN provides improved merging within the corresponding cell types across batches, leading to reduced differentiation across batches over MNN, Seurat v3 and LIGER. Furthermore, SMNN retains more cell-type-specific features, partially manifested by differentially expressed genes identified between cell types after SMNN correction being biologically more relevant, with precision improving by up to 841.0%. [ABSTRACT FROM AUTHOR]

Recent advances in single-cell RNA sequencing (scRNA-seq) enable characterization of transcriptomic profiles with single-cell resolution and circumvent averaging artifacts associated with traditional bulk RNA sequencing (RNA-seq) data. Here, we propose SCDC, a deconvolution method for bulk RNA-seq that leverages cell-type specific gene expression profiles from multiple scRNA-seq reference datasets. SCDC adopts an ENSEMBLE method to integrate deconvolution results from different scRNA-seq datasets that are produced in different laboratories and at different times, implicitly addressing the problem of batch-effect confounding. SCDC is benchmarked against existing methods using both in silico generated pseudo-bulk samples and experimentally mixed cell lines, whose known cell-type compositions serve as ground truths. We show that SCDC outperforms existing methods with improved accuracy of cell-type decomposition under both settings. To illustrate how the ENSEMBLE framework performs in complex tissues under different scenarios, we further apply our method to a human pancreatic islet dataset and a mouse mammary gland dataset. SCDC returns results that are more consistent with experimental designs and that reproduce more significant associations between cell-type proportions and measured phenotypes. [ABSTRACT FROM AUTHOR]