Assessing the impact of transcriptomics data analysis pipelines on downstream functional enrichment results.

Transcriptomics is widely used to assess the state of biological systems. There are many tools for the different steps, such as normalization, differential expression, and enrichment. While numerous studies have examined the impact of method choices on differential expression results, little attention has been paid to their effects on further downstream functional analysis, which typically provides the basis for interpretation and follow-up experiments. To address this, we introduce FLOP, a comprehensive nextflow-based workflow combining methods to perform end-to-end analyses of transcriptomics data. We illustrate FLOP on datasets ranging from end-stage heart failure patients to cancer cell lines. We discovered effects not noticeable at the gene-level, and observed that not filtering the data had the highest impact on the correlation between pipelines in the gene set space. Moreover, we performed three benchmarks to evaluate the 12 pipelines included in FLOP, and confirmed that filtering is essential in scenarios of expected moderate-to-low biological signal. Overall, our results underscore the impact of carefully evaluating the consequences of the choice of preprocessing methods on downstream enrichment analyses. We envision FLOP as a valuable tool to measure the robustness of functional analyses, ultimately leading to more reliable and conclusive biological findings.

Complementing Cell Taxonomies with a Multicellular Analysis of Tissues.

The application of single-cell molecular profiling coupled with spatial technologies has enabled charting of cellular heterogeneity in reference tissues and in disease. This new wave of molecular data has highlighted the expected diversity of single-cell dynamics upon shared external queues and spatial organizations. However, little is known about the relationship between single-cell heterogeneity and the emergence and maintenance of robust multicellular processes in developed tissues and its role in (patho)physiology. Here, we present emerging computational modeling strategies that use increasingly available large-scale cross-condition single-cell and spatial datasets to study multicellular organization in tissues and complement cell taxonomies. This perspective should enable us to better understand how cells within tissues collectively process information and adapt synchronized responses in disease contexts and to bridge the gap between structural changes and functions in tissues.

LIANA+ provides an all-in-one framework for cell-cell communication inference.

The growing availability of single-cell and spatially resolved transcriptomics has led to the development of many approaches to infer cell-cell communication, each capturing only a partial view of the complex landscape of intercellular signalling. Here we present LIANA+, a scalable framework built around a rich knowledge base to decode coordinated inter- and intracellular signalling events from single- and multi-condition datasets in both single-cell and spatially resolved data. By extending and unifying established methodologies, LIANA+ provides a comprehensive set of synergistic components to study cell-cell communication via diverse molecular mediators, including those measured in multi-omics data. LIANA+ is accessible at https://github.com/saezlab/liana-py with extensive vignettes ( https://liana-py.readthedocs.io/ ) and provides an all-in-one solution to intercellular communication inference.

Multicellular factor analysis of single-cell data for a tissue-centric understanding of disease.

Biomedical single-cell atlases describe disease at the cellular level. However, analysis of this data commonly focuses on cell-type-centric pairwise cross-condition comparisons, disregarding the multicellular nature of disease processes. Here, we propose multicellular factor analysis for the unsupervised analysis of samples from cross-condition single-cell atlases and the identification of multicellular programs associated with disease. Our strategy, which repurposes group factor analysis as implemented in multi-omics factor analysis, incorporates the variation of patient samples across cell-types or other tissue-centric features, such as cell compositions or spatial relationships, and enables the joint analysis of multiple patient cohorts, facilitating the integration of atlases. We applied our framework to a collection of acute and chronic human heart failure atlases and described multicellular processes of cardiac remodeling, independent to cellular compositions and their local organization, that were conserved in independent spatial and bulk transcriptomics datasets. In sum, our framework serves as an exploratory tool for unsupervised analysis of cross-condition single-cell atlases and allows for the integration of the measurements of patient cohorts across distinct data modalities.