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Background Despite therapeutic advances, metastatic melanoma, and particularly brain metastasis (MBM), remains a lethal burden for patients. Existing single-cell studies offer a more detailed view of melanoma and its microenvironment, which is crucial to improve diagnosis and treatment. Results We here present a computational reanalysis of single-nucleus data comparing 15 MBM and 10 extracranial melanoma metastases (ECM), considering recent best practice recommendations. We used cell type-specific pseudobulking and omit imputation during patient integration to gain complementary insights. Interestingly, our analysis revealed high homogeneity in tumor cell expression profiles within and between MBM and ECM. However, MBM displayed even higher homogeneity but a more flexible energy metabolism, suggesting a specific metastatic adaptation to the putatively more restricted brain microenvironment. While tumor cells were homogeneous, the metastasis microenvironment, especially lymphocytes and related immune-tumor interaction pathways, exhibited greater divergence between MBM and ECM. Overall, this suggests that major differences between MBM and ECM are potentially driven by variations in their microenvironment. Finally, a comparison of single-cell data to previous bulk studies, including their deconvoluted putative cell types, showed significant differences, potentially causing divergent conclusions. Conclusion Our study contributed to refine the understanding of differences between MBM and ECM, suggesting these are potentially more influenced by their local microenvironments. Future research and therapies could possibly focus on the metabolic flexibility of melanoma brain metastases and patient-specific immune pathway alterations.

Background The analysis of single-cell RNA sequencing (scRNAseq) data plays an important role in understanding the intrinsic and extrinsic cellular processes in biological and biomedical research. One significant effort in this area is the detection of differentially expressed (DE) genes. scRNAseq data, however, are highly heterogeneous and have a large number of zero counts, which introduces challenges in detecting DE genes. Addressing these challenges requires employing new approaches beyond the conventional ones, which are based on a nonzero difference in average expression. Several methods have been developed for differential gene expression analysis of scRNAseq data. To provide guidance on choosing an appropriate tool or developing a new one, it is necessary to evaluate and compare the performance of differential gene expression analysis methods for scRNAseq data. Results In this study, we conducted a comprehensive evaluation of the performance of eleven differential gene expression analysis software tools, which are designed for scRNAseq data or can be applied to them. We used simulated and real data to evaluate the accuracy and precision of detection. Using simulated data, we investigated the effect of sample size on the detection accuracy of the tools. Using real data, we examined the agreement among the tools in identifying DE genes, the run time of the tools, and the biological relevance of the detected DE genes. Conclusions In general, agreement among the tools in calling DE genes is not high. There is a trade-off between true-positive rates and the precision of calling DE genes. Methods with higher true positive rates tend to show low precision due to their introducing false positives, whereas methods with high precision show low true positive rates due to identifying few DE genes. We observed that current methods designed for scRNAseq data do not tend to show better performance compared to methods designed for bulk RNAseq data. Data multimodality and abundance of zero read counts are the main characteristics of scRNAseq data, which play important roles in the performance of differential gene expression analysis methods and need to be considered in terms of the development of new methods.

Recent advances in single-cell sequencing technologies offer an opportunity to explore cell–cell communication in tissues systematically and with reduced bias. A key challenge is integrating known molecular interactions and measurements into a framework to identify and analyze complex cell–cell communication networks. Previously, we developed a computational tool, named CellChat, that infers and analyzes cell–cell communication networks from single-cell transcriptomic data within an easily interpretable framework. CellChat quantifies the signaling communication probability between two cell groups using a simplified mass-action-based model, which incorporates the core interaction between ligands and receptors with multisubunit structure along with modulation by cofactors. Importantly, CellChat performs a systematic and comparative analysis of cell–cell communication using a variety of quantitative metrics and machine-learning approaches. CellChat v2 is an updated version that includes additional comparison functionalities, an expanded database of ligand–receptor pairs along with rich functional annotations, and an Interactive CellChat Explorer. Here we provide a step-by-step protocol for using CellChat v2 on single-cell transcriptomic data, including inference and analysis of cell–cell communication from one dataset and identification of altered intercellular communication, signals and cell populations from different datasets across biological conditions. The R implementation of CellChat v2 toolkit and its tutorials together with the graphic outputs are available at https://github.com/jinworks/CellChat . This protocol typically takes ~5 min depending on dataset size and requires a basic understanding of R and single-cell data analysis but no specialized bioinformatics training for its implementation. Key points CellChat is a software package for systematic inference, quantitative analysis and intuitive visualization of cell–cell communication in an easily interpretable way from single-cell transcriptomic data; it also enables comparative analysis of intercellular communication across different conditions. CellChat v2 is an updated version that includes additional functionalities for comparative analysis and an expanded database of ligand–receptor pairs along with rich functional annotations. CellChat enables systematic inference, quantitative analysis and intuitive visualization of cell–cell communication from single-cell transcriptomic data, as well as comparative analysis of intercellular communication across biological conditions.

Malignant tumors have increasing morbidity and high mortality, and their occurrence and development is a complicate process. The development of sequencing technologies enabled us to gain a better understanding of the underlying genetic and molecular mechanisms in tumors. In recent years, the spatial transcriptomics sequencing technologies have been developed rapidly and allow the quantification and illustration of gene expression in the spatial context of tissues. Compared with the traditional transcriptomics technologies, spatial transcriptomics technologies not only detect gene expression levels in cells, but also inform the spatial location of genes within tissues, cell composition of biological tissues, and interaction between cells. Here we summarize the development of spatial transcriptomics technologies, spatial transcriptomics tools and its application in cancer research. We also discuss the limitations and challenges of current spatial transcriptomics approaches, as well as future development and prospects.

The recent advent of genome-scale imaging has enabled single-cell omics analysis in a spatially resolved manner in intact cells and tissues. These advances allow gene expression profiling of individual cells, and hence in situ identification and spatial mapping of cell types, in complex tissues. The high spatial resolution of these approaches further allows determination of the spatial organizations of the genome and transcriptome inside cells, both of which are key regulatory mechanisms for gene expression.

Background Changes in spatial chromatin interactions are now emerging as a unifying mechanism orchestrating the regulation of gene expression. Hi-C sequencing technology allows insight into chromatin interactions on a genome-wide scale. However, Hi-C data contains many DNA sequence- and technology-driven biases. These biases prevent effective comparison of chromatin interactions aimed at identifying genomic regions differentially interacting between, e.g., disease-normal states or different cell types. Several methods have been developed for normalizing individual Hi-C datasets. However, they fail to account for biases between two or more Hi-C datasets , hindering comparative analysis of chromatin interactions. Results We developed a simple and effective method, HiCcompare, for the joint normalization and differential analysis of multiple Hi-C datasets. The method introduces a distance-centric analysis and visualization of the differences between two Hi-C datasets on a single plot that allows for a data-driven normalization of biases using locally weighted linear regression (loess). HiCcompare outperforms methods for normalizing individual Hi-C datasets and methods for differential analysis (diffHiC, FIND) in detecting a priori known chromatin interaction differences while preserving the detection of genomic structures, such as A/B compartments. Conclusions HiCcompare is able to remove between-dataset bias present in Hi-C matrices. It also provides a user-friendly tool to allow the scientific community to perform direct comparisons between the growing number of pre-processed Hi-C datasets available at online repositories. HiCcompare is freely available as a Bioconductor R package https://bioconductor.org/packages/HiCcompare/ .

Background Deconvolution analyses have been widely used to track compositional alterations of cell types in gene expression data. Although a large number of novel methods have been developed, due to a lack of understanding of the effects of modeling assumptions and tuning parameters, it is challenging for researchers to select an optimal deconvolution method suitable for the targeted biological conditions. Results To systematically reveal the pitfalls and challenges of deconvolution analyses, we investigate the impact of several technical and biological factors including simulation model, quantification unit, component number, weight matrix, and unknown content by constructing three benchmarking frameworks. These frameworks cover comparative analysis of 11 popular deconvolution methods under 1766 conditions. Conclusions We provide new insights to researchers for future application, standardization, and development of deconvolution tools on RNA-seq data.

Analysis of gene expression data using genome-wide microarrays is a technique often used in genomic studies to find coexpression patterns and locate groups of co-transcribed genes. However, most studies done at global \"omic\" scale are not focused on human samples and when they correspond to human very often include heterogeneous datasets, mixing normal with disease-altered samples. Moreover, the technical noise present in genome-wide expression microarrays is another well reported problem that many times is not addressed with robust statistical methods, and the estimation of errors in the data is not provided. Human genome-wide expression data from a controlled set of normal-healthy tissues is used to build a confident human gene coexpression network avoiding both pathological and technical noise. To achieve this we describe a new method that combines several statistical and computational strategies: robust normalization and expression signal calculation; correlation coefficients obtained by parametric and non-parametric methods; random cross-validations; and estimation of the statistical accuracy and coverage of the data. All these methods provide a series of coexpression datasets where the level of error is measured and can be tuned. To define the errors, the rates of true positives are calculated by assignment to biological pathways. The results provide a confident human gene coexpression network that includes 3327 gene-nodes and 15841 coexpression-links and a comparative analysis shows good improvement over previously published datasets. Further functional analysis of a subset core network, validated by two independent methods, shows coherent biological modules that share common transcription factors. The network reveals a map of coexpression clusters organized in well defined functional constellations. Two major regions in this network correspond to genes involved in nuclear and mitochondrial metabolism and investigations on their functional assignment indicate that more than 60% are house-keeping and essential genes. The network displays new non-described gene associations and it allows the placement in a functional context of some unknown non-assigned genes based on their interactions with known gene families. The identification of stable and reliable human gene to gene coexpression networks is essential to unravel the interactions and functional correlations between human genes at an omic scale. This work contributes to this aim, and we are making available for the scientific community the validated human gene coexpression networks obtained, to allow further analyses on the network or on some specific gene associations. The data are available free online at http://bioinfow.dep.usal.es/coexpression/.

Background Effective identification of differentially expressed genes (DEGs) has been challenging for single-cell RNA sequencing (scRNA-seq) profiles. Many existing algorithms have high false positive rates (FPRs) and often fail to identify weak biological signals. Results We present a novel method for identifying DEGs in scRNA-seq data called RankCompV3. It is based on the comparison of relative expression orderings (REOs) of gene pairs which are determined by comparing the expression levels of a pair of genes in a set of single-cell profiles. The numbers of genes with consistently higher or lower expression levels than the gene of interest are counted in two groups in comparison, respectively, and the result is tabulated in a 3 × 3 contingency table which is tested by McCullagh’s method to determine if the gene is dysregulated. In both simulated and real scRNA-seq data, RankCompV3 tightly controlled the FPR and demonstrated high accuracy, outperforming 11 other common single-cell DEG detection algorithms. Analysis with either regular single-cell or synthetic pseudo-bulk profiles produced highly concordant DEGs with the ground-truth. In addition, RankCompV3 demonstrates higher sensitivity to weak biological signals than other methods. The algorithm was implemented using Julia and can be called in R. The source code is available at https://github.com/pathint/RankCompV3.jl . Conclusions The REOs-based algorithm is a valuable tool for analyzing single-cell RNA profiles and identifying DEGs with high accuracy and sensitivity. Key points RankCompV3 is a method for identifying differentially expressed genes (DEGs) in either bulk or single-cell RNA transcriptomics. It is based on the counts of relative expression orderings (REOs) of gene pairs in the two groups. The contingency tables are tested using McCullagh’s method. RankCompV3 has comparable or better performance than that of other conventional methods. It has been shown to be effective in identifying DEGs in both single-cell and pseudo-bulk profiles. Pseudo-bulk method is implemented in RankCompV3, which allows the method to achieve higher computational efficiency and improves the concordance with the bulk ground-truth. RankCompV3 is effective in identifying functionally relevant DEGs in weak-signal datasets. The method is not biased towards highly expressed genes.