SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms.

SQANTI3 is a tool designed for the quality control, curation and annotation of long-read transcript models obtained with third-generation sequencing technologies. Leveraging its annotation framework, SQANTI3 calculates quality descriptors of transcript models, junctions and transcript ends. With this information, potential artifacts can be identified and replaced with reliable sequences. Furthermore, the integrated functional annotation feature enables subsequent functional iso-transcriptomics analyses.

SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms.

The emergence of long-read RNA sequencing (lrRNA-seq) has provided an unprecedented opportunity to analyze transcriptomes at isoform resolution. However, the technology is not free from biases, and transcript models inferred from these data require quality control and curation. In this study, we introduce SQANTI3, a tool specifically designed to perform quality analysis on transcriptomes constructed using lrRNA-seq data. SQANTI3 provides an extensive naming framework to describe transcript model diversity in comparison to the reference transcriptome. Additionally, the tool incorporates a wide range of metrics to characterize various structural properties of transcript models, such as transcription start and end sites, splice junctions, and other structural features. These metrics can be utilized to filter out potential artifacts. Moreover, SQANTI3 includes a Rescue module that prevents the loss of known genes and transcripts exhibiting evidence of expression but displaying low-quality features. Lastly, SQANTI3 incorporates IsoAnnotLite, which enables functional annotation at the isoform level and facilitates functional iso-transcriptomics analyses. We demonstrate the versatility of SQANTI3 in analyzing different data types, isoform reconstruction pipelines, and sequencing platforms, and how it provides novel biological insights into isoform biology. The SQANTI3 software is available at https://github.com/ConesaLab/SQANTI3 .

acorde unravels functionally interpretable networks of isoform co-usage from single cell data.

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 .

Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells.

Retinitis pigmentosa (RP) is a rare, progressive disease that affects photoreceptors and retinal pigment epithelial (RPE) cells with blindness as a final outcome. Despite high medical and social impact, there is currently no therapeutic options to slow down the progression of or cure the disease. The development of effective therapies was largely hindered by high genetic heterogeneity, inaccessible disease tissue, and unfaithful model organisms. The fact that components of ubiquitously expressed splicing factors lead to the retina-specific disease is an additional intriguing question. Herein, we sought to correlate the retinal cell-type-specific disease phenotype with the splicing profile shown by a patient with autosomal recessive RP, caused by a mutation in pre-mRNA splicing factor 8 (PRPF8). In order to get insight into the role of PRPF8 in homeostasis and disease, we capitalize on the ability to generate patient-specific RPE cells and reveal differentially expressed genes unique to RPE cells. We found that spliceosomal complex and ribosomal functions are crucial in determining cell-type specificity through differential expression and alternative splicing (AS) and that PRPF8 mutation causes global changes in splice site selection and exon inclusion that particularly affect genes involved in these cellular functions. This finding corroborates the hypothesis that retinal tissue identity is conferred by a specific splicing program and identifies retinal AS events as a framework toward the design of novel therapeutic opportunities.

Undisclosed, unmet and neglected challenges in multi-omics studies.

Multi-omics approaches have become a reality in both large genomics projects and small laboratories. However, the multi-omics research community still faces a number of issues that have either not been sufficiently discussed or for which current solutions are still limited. In this Perspective, we elaborate on these limitations and suggest points of attention for future research. We finally discuss new opportunities and challenges brought to the field by the rapid development of single-cell high-throughput molecular technologies.

tappAS: a comprehensive computational framework for the analysis of the functional impact of differential splicing.

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.

Single-cell RNAseq for the study of isoforms-how is that possible?

Single-cell RNAseq and alternative splicing studies have recently become two of the most prominent applications of RNAseq. However, the combination of both is still challenging, and few research efforts have been dedicated to the intersection between them. Cell-level insight on isoform expression is required to fully understand the biology of alternative splicing, but it is still an open question to what extent isoform expression analysis at the single-cell level is actually feasible. Here, we establish a set of four conditions that are required for a successful single-cell-level isoform study and evaluate how these conditions are met by these technologies in published research.

Delineating biological and technical variance in single cell expression data.

Single cell transcriptomics is becoming a common technique to unravel new biological phenomena whose functional significance can only be understood in the light of differences in gene expression between single cells. The technology is still in its early days and therefore suffers from many technical challenges. This review discusses the continuous effort to identify and systematically characterise various sources of technical variability in single cell expression data and the need to further develop experimental and computational tools and resources to help deal with it.