Recent advances in long-read sequencing solve inaccuracies in alternative transcript identification of full-length transcripts in short-read RNA-Seq data, which encourages the development of methods for isoform-centered functional analysis. Here, we present tappAS, the first framework to enable a comprehensive Functional Iso-Transcriptomics (FIT) analysis, which is effective at revealing the functional impact of context-specific post-transcriptional regulation. tappAS uses isoform-resolved annotation of coding and non-coding functional domains, motifs, and sites, in combination with novel analysis methods to interrogate different aspects of the functional readout of transcript variants and isoform regulation. tappAS software and documentation are available at https://app.tappas.org.

Traditionally, the functional analysis of gene expression data has used pathway and network enrichment algorithms. These methods are usually gene rather than transcript centric and hence fall short to unravel functional roles associated to posttranscriptional regulatory mechanisms such as Alternative Splicing (AS) and Alternative PolyAdenylation (APA), jointly referred here as Alternative Transcript Processing (AltTP). Moreover, short-read RNA-seq has serious limitations to resolve full-length transcripts, further complicating the study of isoform expression. Recent advances in long-read sequencing open exciting opportunities for studying isoform biology and function. However, there are no established bioinformatics methods for the functional analysis of isoform-resolved transcriptomics data to fully leverage these technological advances. Here we present a novel framework for Functional Iso-Transcriptomics analysis (FIT). This framework uses a rich isoform-level annotation database of functional domains, motifs and sites -both coding and noncoding-and introduces novel analysis methods to interrogate different aspects of the functional relevance of isoform complexity. The Functional Diversity Analysis (FDA) evaluates the variability at the inclusion/exclusion of functional domains across annotated transcripts of the same gene. Parameters can be set to evaluate if AltTP partially or fully disrupts functional elements. FDA is a measure of the potential of a multiple isoform transcriptome to have a functional impact. By combining these functional labels with expression data, the Differential Analysis Module evaluates the relative contribution of transcriptional (i.e. gene level) and post-transcriptional (i.e. transcript/protein levels) regulation on the biology of the system. Measures of inclusion of NLS, transmembrane domains or DNA binding motifs, for example.Some of these findings were experimentally validated by others and us.In summary, we propose a novel framework for the functional analysis of transcriptomes at isoform resolution. We anticipate the tappAS tool will be an important resource for the adoption of the Functional Iso-Transcriptomics analysis by functional genomics community.

Understanding the cause of sex disparities in COVID-19 outcomes is a major challenge. We investigate sex hormone levels and their association with outcomes in COVID-19 patients, stratified by sex and age. This observational, retrospective, cohort study included 138 patients aged 18 years or older with COVID-19, hospitalized in Italy between February 1 and May 30, 2020. The association between sex hormones (testosterone, estradiol, progesterone, dehydroepiandrosterone) and outcomes (ARDS, severe COVID-19, in-hospital mortality) was explored in 120 patients aged 50 years and over. STROBE checklist was followed. The median age was 73.5 years [IQR 61, 82]; 55.8% were male. In older males, testosterone was lower if ARDS and severe COVID-19 were reported than if not (3.6 vs. 5.3 nmol/L, p =0.0378 and 3.7 vs. 8.5 nmol/L, p =0.0011, respectively). Deceased males had lower testosterone (2.4 vs. 4.8 nmol/L, p =0.0536) and higher estradiol than survivors (40 vs. 24 pg/mL, p = 0.0006). Testosterone was negatively associated with ARDS (OR 0.849 [95% CI 0.734, 0.982]), severe COVID-19 (OR 0.691 [95% CI 0.546, 0.874]), and in-hospital mortality (OR 0.742 [95% CI 0.566, 0.972]), regardless of potential confounders, though confirmed only in the regression model on males. Higher estradiol was associated with a higher probability of death (OR 1.051 [95% CI 1.018, 1.084]), confirmed in both sex models. In males, higher testosterone seems to be protective against any considered outcome. Higher estradiol was associated with a higher probability of death in both sexes.

PaintOmics is a web server for the integrative analysis and visualisation of multi-omics datasets using biological pathway maps. PaintOmics 4 has several notable updates that improve and extend analyses. Three pathway databases are now supported: KEGG, Reactome and MapMan, providing more comprehensive pathway knowledge for animals and plants. New metabolite analysis methods fill gaps in traditional pathway-based enrichment methods. The metabolite hub analysis selects compounds with a high number of significant genes in their neighbouring network, suggesting regulation by gene expression changes. The metabolite class activity analysis tests the hypothesis that a metabolic class has a higher-than-expected proportion of significant elements, indicating that these compounds are regulated in the experiment. Finally, PaintOmics 4 includes a regulatory omics module to analyse the contribution of trans-regulatory layers (microRNA and transcription factors, RNA-binding proteins) to regulate pathways. We show the performance of PaintOmics 4 on both mouse and plant data to highlight how these new analysis features provide novel insights into regulatory biology. PaintOmics 4 is available at https://paintomics.org/.

Alternative splicing (AS) is a highly-regulated post-transcriptional mechanism known to modulate isoform expression within genes and contribute to cell-type identity. However, the extent to which alternative isoforms establish co-expression networks that may be relevant in cellular function has not been explored yet. Here, we present acorde, a pipeline that successfully leverages bulk long reads and single-cell data to confidently detect alternative isoform co-expression relationships. To achieve this, we develop and validate percentile correlations, an innovative approach that overcomes data sparsity and yields accurate co-expression estimates from single-cell data. Next, acorde uses correlations to cluster co-expressed isoforms into a network, unraveling cell type-specific alternative isoform usage patterns. By selecting same-gene isoforms between these clusters, we subsequently detect and characterize genes with co-differential isoform usage (coDIU) across cell types. Finally, we predict functional elements from long read-defined isoforms and provide insight into biological processes, motifs, and domains potentially controlled by the coordination of post-transcriptional regulation. The code for acorde is available at https://github.com/ConesaLab/acorde.

Alternative splicing (AS) is a highly-regulated post-transcriptional mechanism known to modulate isoform expression within genes and contribute to cell-type identity. However, the extent to which alternative isoforms establish co-expression networks that may relevant in cellular function has not been explored yet. Here, we present acorde, a pipeline that successfully leverages bulk long reads and single-cell data to confidently detect alternative isoform co-expression relationships. To achieve this, we developed and validated percentile correlations, a novel approach that overcomes data sparsity and yields accurate co-expression estimates from single-cell data. Next, acorde uses correlations to cluster co-expressed isoforms into a network, unraveling cell type-specific alternative isoform usage patterns. By selecting same-gene isoforms between these clusters, we subsequently detect and characterize genes with co-differential isoform usage (coDIU) across neural cell types. Finally, we predict functional elements from long read-defined isoforms and provide insight into biological processes, motifs and domains potentially controlled by the coordination of post-transcriptional regulation.