Alternative pre-mRNA splicing generates functionally distinct transcripts from the same gene and is involved in the control of multiple cellular processes, with its dysregulation being associated with a variety of pathologies. The advent of next-generation sequencing has enabled global studies of alternative splicing in different physiological and disease contexts. However, current bioinformatics tools for alternative splicing analysis from RNA-seq data are not user-friendly, disregard available exon-exon junction quantification or have limited downstream analysis features.
To overcome such limitations, University of Lisbon researchers have developed psichomics, an R package with an intuitive graphical interface for alternative splicing quantification and downstream dimensionality reduction, differential splicing and gene expression and survival analyses based on The Cancer Genome Atlas, the Genotype-Tissue Expression project, the Sequence Read Archive project and user-provided data. These integrative analyses can also incorporate clinical and molecular sample-associated features. The researchers successfully used psichomics in a laptop to reveal alternative splicing signatures specific to stage I breast cancer and associated novel putative prognostic factors.
Alternative splicing analyses on tumour stage I and normal breast cancer samples from TCGA
(A, B) PCA on PSI levels from tumour stage I and normal breast cancer samples; score (A) and loading (B) plots. The loading plot depicts the projection of splicing events on the two first principal components, with selected events labelled with their cognate gene symbol. The bubble size in panel B represents the relative contribution of each alternative splicing event to the selected principal components. (C) Volcano plot of differential splicing analysis performed between tumour stage I and normal breast cancer samples using the Wilcoxon rank-sum test with Benjamini–Hochberg (FDR) adjustment for multiple testing. Significantly differentially spliced events (|Δ median PSI| ≥ 0.1 and FDR ≤ 0.01) are highlighted in orange, with selected events with putative prognostic value depicted in purple. (D) One such event is an UHRF2 skipped exon, whose PSI distributions in tumour stage I and normal samples are depicted in the density plot (left), whereas its prognostic value is illustrated by the Kaplan–Meier survival curves (right; patients separated by a PSI cut-off of 0.09). (E) Protein domain disrupted by UHRF2 exon inclusion. UHRF2 transcripts in blue, UHRF2 exon 10 in green and UniProt domains in red. All images were retrieved from psichomics as is, with the exception of the gene symbol overlay in B and C, the FDR label and the arrow highlighting the PSI cut-off in D and panel E.
Availability -psichomics is available at: https://bioconductor.org/packages/release/bioc/html/psichomics.html