ScisorWiz: visualizing differential isoform expression in single-cell long-read data

Stein, Alexander N.; Joglekar, Anoushka; Poon, Chi-Lam; Tilgner, Hagen U

doi:10.1093/bioinformatics/btac340

Cited by 16 publications

(16 citation statements)

References 23 publications

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“…(d) Distribution of % exons significant of a down-sampling experiment which selected for 20 reads per exon and 100 exons per cell type for 100 iterations. (e) Cell-type resolved single-cell long reads for NFE2L1 gene plotted with ScISOrWiz 2 . Top 2 tracks: L3-5/L6 IT_ RORB in PFC and VIS .…”

Section: Resultsmentioning

confidence: 99%

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer’s Disease

Hu,

Foord,

Hsu

et al. 2024

Preprint

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Multimodal measurements have become widespread in genomics, however measuring open chromatin accessibility and splicing simultaneously in frozen brain tissues remains unconquered. Hence, we devised Single-Cell-ISOform-RNA sequencing coupled with the Assay-for-Transposase-Accessible-Chromatin (ScISOr-ATAC). We utilized ScISOr-ATAC to assess whether chromatin and splicing alterations in the brain convergently affect the same cell types or divergently different ones. We applied ScISOr-ATAC to three major conditions: comparing (i) the Rhesus macaque (Macaca mulatta) prefrontal cortex (PFC) and visual cortex (VIS), (ii) cross species divergence of Rhesus macaque versus human PFC, as well as (iii) dysregulation in Alzheimer's disease in human PFC. We found that among cortical-layer biased excitatory neuron subtypes, splicing is highly brain-region specific for L3-5/L6 IT_RORB neurons, moderately specific in L2-3 IT_CUX2.RORB neurons and unspecific in L2-3 IT_CUX2 neurons. In contrast, at the chromatin level, L2-3 IT_CUX2.RORB neurons show the highest brain-region specificity compared to other subtypes. Likewise, when comparing human and macaque PFC, strong evolutionary divergence on one molecular modality does not necessarily imply strong such divergence on another molecular level in the same cell type. Finally, in Alzheimer's disease, oligodendrocytes show convergently high dysregulation in both chromatin and splicing. However, chromatin and splicing dysregulation most strongly affect distinct oligodendrocyte subtypes. Overall, these results indicate that chromatin and splicing can show convergent or divergent results depending on the performed comparison, justifying the need for their concurrent measurement to investigate complex systems. Taken together, ScISOr-ATAC allows for the characterization of single-cell splicing and chromatin patterns and the comparison of sample groups in frozen brain samples.

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Section: Resultsmentioning

confidence: 99%

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer’s Disease

Hu,

Foord,

Hsu

et al. 2024

Preprint

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“…In VIS, Dnm2 has exons in G5 and G8 and therefore exemplifies repetitive developmental changes in cell-type variability. The ScisorWiz 74 plot shows Dnm2 leveraging four alternative exons to produce 6 complex isoforms. In excitatory neurons at P14, two mutually exclusive exons (Exons A,B) define two major isoforms, the second of which dominates at P21 and P28.…”

Section: Marker Exons Delineate Cell-type and Brain-region Specific S...mentioning

confidence: 99%

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

Joglekar

Zhang³

et al. 2023

Preprint

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RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

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“…3e). In order to visualize this on a case-by-case basis, we plotted reads from both assays with ScisorWiz (Stein et al, 2022). Amongst the 5’-longer genes, we chose the PABPC1 gene as an example, with over 100 reads in both libraries.…”

Section: Introductionmentioning

confidence: 99%

Comparing 10x Genomics single-cell 3’ and 5’ assay in short-and long-read sequencing

Hsu

Jarroux

Joglekar

et al. 2022

Preprint

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Barcoding strategies are fundamental to droplet-based single-cell sequencing, and understanding the biases and caveats between approaches is essential. Here, we comprehensively evaluated both short and long reads of the cDNA obtained through the two marketed approaches from 10x Genomics, the "3' assay" and the "5' assay", which attach barcodes at different ends of the mRNA molecule. Although the barcode detection, cell-type identification, and gene expression profile are similar in both assays, the 5' assay captured more exonic molecules and fewer intronic molecules compared to the 3' assay. We found that 13.7% of genes sequenced have longer average read lengths and are more complete (spanning both polyA-site and TSS) in the long reads from the 5' assay compared to the 3' assay. These genes are characterized by long average transcript length, high intron number, and low expression overall. Despite these differences, cell-type-specific isoform profiles observed from the two assays remain highly correlated. This study provides a benchmark for choosing the single-cell assay for the intended research question, and insights regarding platform-specific biases to be mindful of when analyzing data, particularly across samples and technologies.

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ScisorWiz: visualizing differential isoform expression in single-cell long-read data

Cited by 16 publications

References 23 publications

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer’s Disease

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer’s Disease

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

Comparing 10x Genomics single-cell 3’ and 5’ assay in short-and long-read sequencing

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