Spatial isoform sequencing at sub-micrometer single-cell resolution reveals novel patterns of spatial isoform variability in brain cell types Article Swipe
Lieke Michielsen
,
Andrey D. Prjibelski
,
Careen Foord
,
Wen Hu
,
Julien Jarroux
,
Justine Hsu
,
Alexandru I. Tomescu
,
Iman Hajirasouliha
,
Hagen Tilgner
·
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1101/2025.06.25.661563
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1101/2025.06.25.661563
Spatial long-read technologies are becoming increasingly common but lack nanometer- and therefore often single-cell resolution. This leaves the question unanswered of whether spatially variable isoforms represent spatial variability within one cell type or differences in cell-type composition between different regions. Here, we developed Spl-ISO-Seq2 (220nm spot size and 500nm resolution), and the accompanying software packages Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing using 140 million barcodes compared to 80,000 previously. Applying this to the adult mouse brain, we compared spatial variability by examining (a) differential isoform abundance between known brain regions and (b) spatial isoform patterns that do not align with predefined regions. While the former revealed more spatial isoform differences, both approaches identified overlapping hits, e.g., Rps24 in oligodendrocytes. For Snap25, previously known to exhibit spatial isoform variation, we now show that this variability occurs in excitatory neurons. The second approach also uncovered patterns not captured by predefined-region comparisons, e.g., Tnnc1 in excitatory neurons. Furthermore, we show that a surprising number of spatial isoform signals is not driven by cell-type composition alone. Finally, we applied our software to public Visium HD 3’ long-read data to demonstrate its applicability and strong reproducibility across protocols and biological replicates. Taken together, our experimental and analytical methods enrich spatial transcriptomics with a so-far elusive isoform view of spatial variation for individual cell types.
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- Type
- preprint
- Language
- en
- Landing Page
- https://doi.org/10.1101/2025.06.25.661563
- https://www.biorxiv.org/content/biorxiv/early/2025/06/25/2025.06.25.661563.full.pdf
- OA Status
- green
- Cited By
- 1
- References
- 56
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4411666668
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Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W4411666668Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1101/2025.06.25.661563Digital Object Identifier
- Title
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Spatial isoform sequencing at sub-micrometer single-cell resolution reveals novel patterns of spatial isoform variability in brain cell typesWork title
- Type
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preprintOpenAlex work type
- Language
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enPrimary language
- Publication year
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2025Year of publication
- Publication date
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2025-06-25Full publication date if available
- Authors
-
Lieke Michielsen, Andrey D. Prjibelski, Careen Foord, Wen Hu, Julien Jarroux, Justine Hsu, Alexandru I. Tomescu, Iman Hajirasouliha, Hagen TilgnerList of authors in order
- Landing page
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https://doi.org/10.1101/2025.06.25.661563Publisher landing page
- PDF URL
-
https://www.biorxiv.org/content/biorxiv/early/2025/06/25/2025.06.25.661563.full.pdfDirect link to full text PDF
- Open access
-
YesWhether a free full text is available
- OA status
-
greenOpen access status per OpenAlex
- OA URL
-
https://www.biorxiv.org/content/biorxiv/early/2025/06/25/2025.06.25.661563.full.pdfDirect OA link when available
- Concepts
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Gene isoform, Micrometer, Focus (optics), Cell, Biology, Computational biology, Neuroscience, Genetics, Physics, Optics, GeneTop concepts (fields/topics) attached by OpenAlex
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1Total citation count in OpenAlex
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2025: 1Per-year citation counts (last 5 years)
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56Number of works referenced by this work
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10Other works algorithmically related by OpenAlex
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| abstract_inverted_index.its | 187 |
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| abstract_inverted_index.500nm | 49 |
| abstract_inverted_index.Here, | 41 |
| abstract_inverted_index.Rps24 | 117 |
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| abstract_inverted_index.Tnnc1 | 151 |
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| abstract_inverted_index.brain | 89 |
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| abstract_inverted_index.hits, | 115 |
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| abstract_inverted_index.mouse | 75 |
| abstract_inverted_index.often | 13 |
| abstract_inverted_index.using | 62 |
| abstract_inverted_index.(220nm | 45 |
| abstract_inverted_index.80,000 | 68 |
| abstract_inverted_index.Visium | 180 |
| abstract_inverted_index.across | 192 |
| abstract_inverted_index.alone. | 172 |
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| abstract_inverted_index.oligodendrocytes. | 119 |
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| corresponding_author_ids | https://openalex.org/A5070938580 |
| countries_distinct_count | 3 |
| institutions_distinct_count | 9 |
| corresponding_institution_ids | https://openalex.org/I205783295, https://openalex.org/I4210097825, https://openalex.org/I4210102457, https://openalex.org/I4387153466 |
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| citation_normalized_percentile.is_in_top_10_percent | True |