Several methods have been developed to computationally predict cell-types for single cell RNA sequencing (scRNAseq) data. As methods are developed, a common problem for investigators has been identifying the best method they should apply to their specific use-case. To address this challenge, we present CHAI (consensus Clustering tHrough similArIty matrix integratIon for single cell-type identification), a wisdom of crowds approach for scRNAseq clustering. CHAI presents two competing methods which aggregate the clustering results from seven state-of-the-art clustering methods: CHAI-AvgSim and CHAI-SNF. CHAI-AvgSim and CHAI-SNF demonstrate superior performance across several benchmarking datasets. Furthermore, both CHAI methods outperform the most recent consensus clustering method, SAME-clustering. We demonstrate CHAI\'s practical use case by identifying a leader tumor cell cluster enriched with CDH3. CHAI provides a platform for multiomic integration, and we demonstrate CHAI-SNF to have improved performance when including spatial transcriptomics data. CHAI overcomes previous limitations by incorporating the most recent and top performing scRNAseq clustering algorithms into the aggregation framework. It is also an intuitive and easily customizable R package where users may add their own clustering methods to the pipeline, or down-select just the ones they want to use for the clustering aggregation. This ensures that as more advanced clustering algorithms are developed, CHAI will remain useful to the community as a generalized framework. CHAI is available as an open source R package on GitHub: https://github.com/lodimk2/chai.

Single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (SRT) have led to groundbreaking advancements in life sciences. To develop bioinformatics tools for scRNA-seq and SRT data and perform unbiased benchmarks, data simulation has been widely adopted by providing explicit ground truth and generating customized datasets. However, the performance of simulation methods under multiple scenarios has not been comprehensively assessed, making it challenging to choose suitable methods without practical guidelines.
We systematically evaluated 49 simulation methods developed for scRNA-seq and/or SRT data in terms of accuracy, functionality, scalability, and usability using 152 reference datasets derived from 24 platforms. SRTsim, scDesign3, ZINB-WaVE, and scDesign2 have the best accuracy performance across various platforms. Unexpectedly, some methods tailored to scRNA-seq data have potential compatibility for simulating SRT data. Lun, SPARSim, and scDesign3-tree outperform other methods under corresponding simulation scenarios. Phenopath, Lun, Simple, and MFA yield high scalability scores but they cannot generate realistic simulated data. Users should consider the trade-offs between method accuracy and scalability (or functionality) when making decisions. Additionally, execution errors are mainly caused by failed parameter estimations and appearance of missing or infinite values in calculations. We provide practical guidelines for method selection, a standard pipeline Simpipe ( https://github.com/duohongrui/simpipe ; https://doi.org/10.5281/zenodo.11178409 ), and an online tool Simsite ( https://www.ciblab.net/software/simshiny/ ) for data simulation.
No method performs best on all criteria, thus a good-yet-not-the-best method is recommended if it solves problems effectively and reasonably. Our comprehensive work provides crucial insights for developers on modeling gene expression data and fosters the simulation process for users.

Intra-tumor heterogeneity compromises the clinical value of transcriptomic classifications of colorectal cancer. We investigated the prognostic effect of transcriptomic heterogeneity and the potential for classifications less vulnerable to heterogeneity in a single-hospital series of 1093 tumor samples from 692 patients, including multiregional samples from 98 primary tumors and 35 primary-metastasis sets. We show that intra-tumor heterogeneity of the consensus molecular subtypes (CMS) is frequent and has poor-prognostic associations independently of tumor microenvironment markers. Multiregional transcriptomics uncover cancer cell-intrinsic and low-heterogeneity signals that recapitulate the intrinsic CMSs proposed by single-cell sequencing. Further subclassification identifies congruent CMSs that explain a larger proportion of variation in patient survival than intra-tumor heterogeneity. Plasticity is indicated by discordant intrinsic phenotypes of matched primary and metastatic tumors. We conclude that multiregional sampling reconciles the prognostic power of tumor classifications from single-cell and bulk transcriptomics in the context of intra-tumor heterogeneity, and phenotypic plasticity challenges the reconciliation of primary and metastatic subtypes.

High heterogeneity and complex interactions of malignant cells in breast cancer has been recognized as a driver of cancer progression and therapeutic failure. However, complete understanding of common cancer cell states and their underlying driver factors remain scarce and challenging. Here, we revealed seven consensus cancer cell states recurring cross patients by integrative analysis of single-cell RNA sequencing data of breast cancer. The distinct biological functions, the subtype-specific distribution, the potential cells of origin and the interrelation of consensus cancer cell states were systematically elucidated and validated in multiple independent datasets. We further uncovered the internal regulons and external cell components in tumor microenvironments, which contribute to the consensus cancer cell states. Using the state-specific signature, we also inferred the abundance of cells with each consensus cancer cell state by deconvolution of large breast cancer RNA-seq cohorts, revealing the association of immune-related state with better survival. Our study provides new insights for the cancer cell state composition and potential therapeutic strategies of breast cancer.

Single-cell RNA sequencing (scRNA-seq) data, annotated by cell type, is useful in a variety of downstream biological applications, such as profiling gene expression at the single-cell level. However, manually assigning these annotations with known marker genes is both time-consuming and subjective.
We present a Graph Convolutional Network (GCN)-based approach to automate the annotation process. Our process builds upon existing labeling approaches, using state-of-the-art tools to find cells with highly confident label assignments through consensus and spreading these confident labels with a semi-supervised GCN. Using simulated data and two scRNA-seq datasets from different tissues, we show that our method improves accuracy over a simple consensus algorithm and the average of the underlying tools. We also compare our method to a nonparametric neighbor majority approach, showing comparable results. We then demonstrate that our GCN method allows for feature interpretation, identifying important genes for cell type classification. We present our completed pipeline, written in PyTorch, as an end-to-end tool for automating and interpreting the classification of scRNA-seq data.
Our code for conducting the experiments in this paper and using our model is available at https://github.com/lewinsohndp/scSHARP.

BACKGROUND: In recent years, the introduction of single-cell RNA sequencing (scRNA-seq) has enabled the analysis of a cell\'s transcriptome at an unprecedented granularity and processing speed. The experimental outcome of applying this technology is a [Formula: see text] matrix containing aggregated mRNA expression counts of M genes and N cell samples. From this matrix, scientists can study how cell protein synthesis changes in response to various factors, for example, disease versus non-disease states in response to a treatment protocol. This technology\'s critical challenge is detecting and accurately recording lowly expressed genes. As a result, low expression levels tend to be missed and recorded as zero - an event known as dropout. This makes the lowly expressed genes indistinguishable from true zero expression and different than the low expression present in cells of the same type. This issue makes any subsequent downstream analysis difficult.
RESULTS: To address this problem, we propose an approach to measure cell similarity using consensus clustering and demonstrate an effective and efficient algorithm that takes advantage of this new similarity measure to impute the most probable dropout events in the scRNA-seq datasets. We demonstrate that our approach exceeds the performance of existing imputation approaches while introducing the least amount of new noise as measured by clustering performance characteristics on datasets with known cell identities.
CONCLUSIONS: ccImpute is an effective algorithm to correct for dropout events and thus improve downstream analysis of scRNA-seq data. ccImpute is implemented in R and is available at https://github.com/khazum/ccImpute .

Thanks to the development of high-throughput sequencing technologies, massive amounts of various biomolecular data have been accumulated to revolutionize the study of genomics and molecular biology. One of the main challenges in analyzing this biomolecular data is to cluster their subtypes into subpopulations to facilitate subsequent downstream analysis. Recently, many clustering methods have been developed to address the biomolecular data. However, the computational methods often suffer from many limitations such as high dimensionality, data heterogeneity and noise.
In our study, we develop a novel Graph-based Multiple Hierarchical Consensus Clustering (GMHCC) method with an unsupervised graph-based feature ranking (FR) and a graph-based linking method to explore the multiple hierarchical information of the underlying partitions of the consensus clustering for multiple types of biomolecular data. Indeed, we first propose to use a graph-based unsupervised FR model to measure each feature by building a graph over pairwise features and then providing each feature with a rank. Subsequently, to maintain the diversity and robustness of basic partitions (BPs), we propose multiple diverse feature subsets to generate several BPs and then explore the hierarchical structures of the multiple BPs by refining the global consensus function. Finally, we develop a new graph-based linking method, which explicitly considers the relationships between clusters to generate the final partition. Experiments on multiple types of biomolecular data including 35 cancer gene expression datasets and eight single-cell RNA-seq datasets validate the effectiveness of our method over several state-of-the-art consensus clustering approaches. Furthermore, differential gene analysis, gene ontology enrichment analysis and KEGG pathway analysis are conducted, providing novel insights into cell developmental lineages and characterization mechanisms.
The source code is available at GitHub: https://github.com/yifuLu/GMHCC. The software and the supporting data can be downloaded from: https://figshare.com/articles/software/GMHCC/17111291.
Supplementary data are available at Bioinformatics online.

Alzheimer\'s disease (AD) is the most common form of dementia, characterized by progressive cognitive impairment and neurodegeneration. Extensive clinical and genomic studies have revealed biomarkers, risk factors, pathways, and targets of AD in the past decade. However, the exact molecular basis of AD development and progression remains elusive. The emerging single-cell sequencing technology can potentially provide cell-level insights into the disease. Here we systematically review the state-of-the-art bioinformatics approaches to analyze single-cell sequencing data and their applications to AD in 14 major directions, including 1) quality control and normalization, 2) dimension reduction and feature extraction, 3) cell clustering analysis, 4) cell type inference and annotation, 5) differential expression, 6) trajectory inference, 7) copy number variation analysis, 8) integration of single-cell multi-omics, 9) epigenomic analysis, 10) gene network inference, 11) prioritization of cell subpopulations, 12) integrative analysis of human and mouse sc-RNA-seq data, 13) spatial transcriptomics, and 14) comparison of single cell AD mouse model studies and single cell human AD studies. We also address challenges in using human postmortem and mouse tissues and outline future developments in single cell sequencing data analysis. Importantly, we have implemented our recommended workflow for each major analytic direction and applied them to a large single nucleus RNA-sequencing (snRNA-seq) dataset in AD. Key analytic results are reported while the scripts and the data are shared with the research community through  GitHub. In summary, this comprehensive review provides insights into various approaches to analyze single cell sequencing data and offers specific guidelines for study design and a variety of analytic directions. The review and the accompanied software tools will serve as a valuable resource for studying cellular and molecular mechanisms of AD, other diseases, or biological systems at the single cell level.

Single-cell transcriptomics can profile thousands of cells in a single experiment and identify novel cell types, states and dynamics in a wide variety of tissues and organisms. Standard experimental protocols and analysis workflows have been developed to create single-cell transcriptomic maps from tissues. This tutorial focuses on how to interpret these data to identify cell types, states and other biologically relevant patterns with the objective of creating an annotated map of cells. We recommend a three-step workflow including automatic cell annotation (wherever possible), manual cell annotation and verification. Frequently encountered challenges are discussed, as well as strategies to address them. Guiding principles and specific recommendations for software tools and resources that can be used for each step are covered, and an R notebook is included to help run the recommended workflow. Basic familiarity with computer software is assumed, and basic knowledge of programming (e.g., in the R language) is recommended.

Single-cell RNA sequencing (scRNA-seq) is a popular and powerful technology that allows you to profile the whole transcriptome of a large number of individual cells. However, the analysis of the large volumes of data generated from these experiments requires specialized statistical and computational methods. Here we present an overview of the computational workflow involved in processing scRNA-seq data. We discuss some of the most common tasks and the tools available for addressing central biological questions. In this article and our companion website ( https://scrnaseq-course.cog.sanger.ac.uk/website/index.html ), we provide guidelines regarding best practices for performing computational analyses. This tutorial provides a hands-on guide for experimentalists interested in analyzing their data as well as an overview for bioinformaticians seeking to develop new computational methods.