The first single cell long-read sequencing dataset of fetal human retina can help elucidate the role of alternative splicing in retinal development and guide the design of transcript-specific gene therapies for retinal regeneration.
Abstract
Retinal development has been extensively studied and key transcriptional regulators that drive fate decisions have been identified for major cell classes. These findings were confirmed and deepened in recent years with the advance of single cell RNA sequencing (Scrase). However, many processes that guide progenitor to postmitotic cell differentiation remain elusive, especially since some genes seem to yield different cell populations without apparent correlation with expression level or timing. Here, differential transcript isoform usage might play a role in diversifying the function of developmental genes. In short-read based scRNAseq, isoforms can only be identified if a read maps to a unique sequence or exon junction. However, due to the sparsity and very short reads, these events are extremely rare. We combined a commercial scRNAseq kit, that produces barcoded, full-length cDNA with Oxford Nanopore Technologies based long-read sequencing to generate the first single cell long-read sequencing dataset of fetal human retina. It can help elucidate the role of alternative splicing in retinal development and guide the design of transcript-specific gene therapies for retinal regeneration.
RNA splicing shapes neuronal identity and disease risk, yet current maps lack the developmental resolution and depth to resolve this complexity. Here, we integrate deep long-read RNA sequencing and proteomics in induced pluripotent stem cell-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half previously unknown) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Population genetics demonstrates that variants affecting novel exons and splice sites are under negative selection, underscoring the potential significance of these isoforms. During neuronal maturation, we observe that autism risk genes undergo dynamic isoform switching, including microexon inclusion and intron retention, that remodel key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between alternative transcript processing events, including transcription start sites, exon splicing, and polyadenylation. Finally, our atlas enables variant reinterpretation in autism, highlighting the value of an isoform-centric view for interpreting pathogenic variation in neurodevelopment. Alternative splicing expands neuronal diversity, but many brain RNA isoforms remain uncharacterized. Here, the authors generate a high‑resolution proteogenomic atlas of human neuron development, uncovering thousands of novel isoforms and revealing dynamic splicing changes in autism risk genes.
N. Xu, Katherine M. Rynard, E. Radley et al.· Nature Communications· 1 citation
Fullscope-seq resolved hundreds of major isoform switches across distinct brain regions and identified DTUs between superficial and deep cortical layers and showed substantial enrichment for neuropsychiatric disorder-associated genes.
Hengxin Liu, Yanhong Hong, Y. Zhang et al.· Nature Methods· 0 citations
Alternative splicing greatly expands the diversity of gene products encoded by the human genome. Single-cell transcriptomic atlases have characterized human cell types through gene-level expression, but short-read sequencing has limited the ability to resolve full-length isoforms and their functional consequences. Here, we present a cross-tissue single-cell long-read isoform atlas spanning 26 human tissues. We identify hundreds of thousands of novel isoforms along with their cell-type-specific usage, and discover that over one-third of expressed isoforms are absent from existing reference databases. We further demonstrate that isoform usage is a structured, measurable axis of cellular identity that is distinct from gene expression. Applying this framework to cellular senescence, we resolve p16INK4a and p14ARF transcripts from the CDKN2A locus in individual cells and uncover cell-type-dependent isoform remodeling associated with the p16INK4a senescence program. This isoform-resolved single-cell atlas offers a versatile framework to dissect the cellular logic of isoform regulation in senescence and beyond.
Madhav Mantri, A. Detweiler, Jaeyoon Lee et al.· bioRxiv· 0 citations
Innate immune responses triggered by Drosophila larval hemocytes have been extensively characterized. However, the full extent of transcriptional and post-transcriptional regulation underlying these processes remains poorly understood. Here, we employed a hybrid sequencing strategy integrating Oxford Nanopore long-read and Illumina short-read sequencing to provide a more comprehensive transcriptome annotation. This enabled discovery of full-length transcripts with novel 5' and 3' boundaries and uncovered 349 previously unannotated long non-coding RNAs highly induced during late stages of wasp infestation. To ensure high confidence transcript models, we further eliminated potential intra-priming artifacts specific to long-read cDNA data. This high-confident full-length transcript models helped to reveal cell type-specific lncRNA markers in lamellocytes and crystal cells by single-cell analyses, which recapitulated hemocyte differentiation trajectories. Notably, RNAi-based depletion of two highly induced lncRNAs impaired lamellocyte formation under wasp infestation, highlighting their functional relevance. Collectively, our findings provide detailed insights into the Drosophila larval immune transcriptomes through the long-read sequencing and highlight the regulatory roles of non-coding RNAs in innate immunity.
Kyung-Tae Lee, Bumsik Cho, Daewon Lee et al.· Genetics· 0 citations
Short-read sequencing-based single-cell transcriptomics represents the current gold standard for studying cellular transcriptomes but remains limited in its ability to resolve full-length transcript isoforms and splicing patterns. Long-read single-cell and single-nucleus RNA sequencing (LR sc/snRNA-seq) enables the transcriptome-wide characterization of full-length isoforms at cellular resolution, yet the relative performance of commercially available workflows remains insufficiently explored. Here, using nuclei extracted from a standardized multi-species benchmark sample and Oxford Nanopore Technologies long-read sequencing, we systematically benchmarked four LR snRNA-seq strategies: 10x Genomics 3’, 10x Genomics 5’, ArgenTag, and Parse Biosciences. Comparing transcriptome features qualitatively and quantitatively, as well as the concordance with matched short-read data and the ability to resolve cellular heterogeneity, we identified substantial method-specific differences in read length and yield, transcript coverage, isoform detection, and recovery of sample-specific biological information, with the 10x Genomics 3’ and 5’ assays emerging as the most balanced approaches for comprehensive isoform-resolved single-nucleus transcriptomics. Altogether, our study provides a systematic assessment of four commercially available workflows for performing LR snRNA-seq and highlights key methodological trade-offs related to distinct library preparation strategies, thus providing practical guidance for future isoform-resolved transcriptome studies at the single-nucleus level.
F. Köhler, Anna Delgado-Tejedor, Maik Zehnsdorf et al.· bioRxiv· 0 citations
Spatial long-read technologies are increasingly common but usually lack single-cell resolution. This leaves unanswered whether spatially variable isoforms reflect variability within one cell type or differences in region-specific cell-type composition. Here, we developed Spl-ISO-Seq2 (500-nm resolution) and accompanying software, Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing of >450 million barcodes versus 80,000 previously. Applying this to the adult mouse brain, we compared differential isoform abundance between known regions and spatial isoform patterns independent of predefined regions. Both identified overlapping hits, for example, Rps24 in oligodendrocytes. For known Snap25 spatial isoform variation, we show that it occurs in excitatory neurons. The region-agnostic approach also uncovered patterns missed by region-based comparisons, for example, for Ighm. Notably, many spatial isoform signals are not driven by cell-type composition alone. Finally, our software is applicable to many spatial and single-cell protocols, demonstrating reproducibility between platforms (for example, Visium HD/Stereo-seq). Overall, our experimental/analytical methods enable a submicron-resolution-isoform view and open avenues for spatial isoform disease research. Spl-ISO-Seq2, Spl-IsoQuant-2 and Spl-IsoFind enable isoform sequencing, barcode calling of >450 million barcodes, and spatially variable isoform detection with high spatial resolution as demonstrated on mouse brain slices.
Lieke Michielsen, Andrey D. Prjibelski, Careen Foord et al.· Nature Methods· 0 citations
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