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granulator is an R package for the cell type deconvolution of heterogeneous tissues based on bulk RNA-seq data or single cell RNA-seq expression profiles. The package provides a unified testing interface to rapidly run and benchmark multiple state-of-the-art deconvolution methods. Data for the deconvolution of peripheral blood mononuclear cells (PBMCs) into individual immune cell types is provided as well.

The VISTA (Visualization and Integrated System for Transcriptomic Analysis) platform streamlines differential expression workflows by wrapping DESeq2 and edgeR into a SummarizedExperiment-based container with consistent metadata. The package includes visualization utilities, MSigDB enrichment helpers, and optional deconvolution support to simplify interactive exploration of RNA-seq experiments.

We present a novel statistical framework for identifying differential distributions in single-cell RNA-sequencing (scRNA-seq) data between treatment conditions by modeling gene expression read counts using generalized linear models (GLMs). We model each gene independently under each treatment condition using error distributions Poisson (P), Negative Binomial (NB), Zero-inflated Poisson (ZIP) and Zero-inflated Negative Binomial (ZINB) with log link function and model based normalization for differences in sequencing depth. Since all four distributions considered in our framework belong to the same family of distributions, we first perform a Kolmogorov-Smirnov (KS) test to select genes belonging to the family of ZINB distributions. Genes passing the KS test will be then modeled using GLMs. Model selection is done by calculating the Bayesian Information Criterion (BIC) and likelihood ratio test (LRT) statistic.

The RNAseqCovarImpute package makes linear model analysis for RNA sequencing read counts compatible with multiple imputation (MI) of missing covariates. A major problem with implementing MI in RNA sequencing studies is that the outcome data must be included in the imputation prediction models to avoid bias. This is difficult in omics studies with high-dimensional data. The first method we developed in the RNAseqCovarImpute package surmounts the problem of high-dimensional outcome data by binning genes into smaller groups to analyze pseudo-independently. This method implements covariate MI in gene expression studies by 1) randomly binning genes into smaller groups, 2) creating M imputed datasets separately within each bin, where the imputation predictor matrix includes all covariates and the log counts per million (CPM) for the genes within each bin, 3) estimating gene expression changes using `limma::voom` followed by `limma::lmFit` functions, separately on each M imputed dataset within each gene bin, 4) un-binning the gene sets and stacking the M sets of model results before applying the `limma::squeezeVar` function to apply a variance shrinking Bayesian procedure to each M set of model results, 5) pooling the results with Rubins’ rules to produce combined coefficients, standard errors, and P-values, and 6) adjusting P-values for multiplicity to account for false discovery rate (FDR). A faster method uses principal component analysis (PCA) to avoid binning genes while still retaining outcome information in the MI models. Binning genes into smaller groups requires that the MI and limma-voom analysis is run many times (typically hundreds). The more computationally efficient MI PCA method implements covariate MI in gene expression studies by 1) performing PCA on the log CPM values for all genes using the Bioconductor `PCAtools` package, 2) creating M imputed datasets where the imputation predictor matrix includes all covariates and the optimum number of PCs to retain (e.g., based on Horn’s parallel analysis or the number of PCs that account for >80% explained variation), 3) conducting the standard limma-voom pipeline with the `voom` followed by `lmFit` followed by `eBayes` functions on each M imputed dataset, 4) pooling the results with Rubins’ rules to produce combined coefficients, standard errors, and P-values, and 5) adjusting P-values for multiplicity to account for false discovery rate (FDR).

Several quantitative and visualized benchmarks for RNA-seq quantification pipelines. Two-condition quantifications for genes, transcripts, junctions or exons by each pipeline with necessary meta information should be organized into numeric matrices in order to proceed the evaluation.

RNA-sense tool compares RNA-seq time curves in two experimental conditions, i.e. wild-type and mutant, and works in three steps. At Step 1, it builds expression profile for each transcript in one condition (i.e. wild-type) and tests if the transcript abundance grows or decays significantly. Dynamic transcripts are then sorted to non-overlapping groups (time profiles) by the time point of switch up or down. At Step 2, RNA-sense outputs the groups of differentially expressed transcripts, which are up- or downregulated in the mutant compared to the wild-type at each time point. At Step 3, Correlations (Fisher's exact test) between the outputs of Step 1 (switch up- and switch down- time profile groups) and the outputs of Step2 (differentially expressed transcript groups) are calculated. The results of the correlation analysis are printed as two-dimensional color plot, with time profiles and differential expression groups at y- and x-axis, respectively, and facilitates the biological interpretation of the data.

Relative transcript abundance has proven to be a valuable tool for understanding the function of genes in biological systems. For the differential analysis of transcript abundance using RNA sequencing data, the negative binomial model is by far the most frequently adopted. However, common methods that are based on a negative binomial model are not robust to extreme outliers, which we found to be abundant in public datasets. So far, no rigorous and probabilistic methods for detection of outliers have been developed for RNA sequencing data, leaving the identification mostly to visual inspection. Recent advances in Bayesian computation allow large-scale comparison of observed data against its theoretical distribution given in a statistical model. Here we propose ppcseq, a key quality-control tool for identifying transcripts that include outlier data points in differential expression analysis, which do not follow a negative binomial distribution. Applying ppcseq to analyse several publicly available datasets using popular tools, we show that from 3 to 10 percent of differentially abundant transcripts across algorithms and datasets had statistics inflated by the presence of outliers.

Principal Component Analysis (PCA) is a very powerful technique that has wide applicability in data science, bioinformatics, and further afield. It was initially developed to analyse large volumes of data in order to tease out the differences/relationships between the logical entities being analysed. It extracts the fundamental structure of the data without the need to build any model to represent it. This 'summary' of the data is arrived at through a process of reduction that can transform the large number of variables into a lesser number that are uncorrelated (i.e. the 'principal components'), while at the same time being capable of easy interpretation on the original data. PCAtools provides functions for data exploration via PCA, and allows the user to generate publication-ready figures. PCA is performed via BiocSingular - users can also identify optimal number of principal components via different metrics, such as elbow method and Horn's parallel analysis, which has relevance for data reduction in single-cell RNA-seq (scRNA-seq) and high dimensional mass cytometry data.

Volcano plots represent a useful way to visualise the results of differential expression analyses. Here, we present a highly-configurable function that produces publication-ready volcano plots. EnhancedVolcano will attempt to fit as many point labels in the plot window as possible, thus avoiding 'clogging' up the plot with labels that could not otherwise have been read. Other functionality allows the user to identify up to 4 different types of attributes in the same plot space via colour, shape, size, and shade parameter configurations.

BreastSubtypeR provides an assumption-aware, multi-method framework for intrinsic molecular subtyping of breast cancer. The package harmonizes several published nearest-centroid (NC) and single-sample predictor (SSP) classifiers, supplies method-specific preprocessing and robust probe-to-gene mapping, and implements a cohort-aware AUTO mode that selectively enables classifiers compatible with the cohort composition. A local Shiny app (iBreastSubtypeR) is included for interactive analyses and to support users without programming experience.