Deciphering the largest disease-associated transcript isoforms in the human neural retina with advanced long-read sequencing approaches
- Merel Stemerdink1,2,
- Tabea Riepe3,4,
- Nick Zomer4,
- Renee Salz3,
- Michael Kwint4,
- Jaap Oostrik1,
- Raoul Timmermans4,
- Barbara Ferrari5,
- Stefano Ferrari5,
- Alfredo Dueñas Rey6,7,
- Emma Delanote6,7,
- Suzanne E. de Bruijn1,4,
- Hannie Kremer1,2,4,
- Susanne Roosing4,
- Frauke Coppieters6,7,8,
- Alexander Hoischen4,9,
- Frans P.M. Cremers4,
- Peter A.C. ‘t Hoen3,
- Erwin van Wijk1,10 and
- Erik de Vrieze1,10
- 1Department of Otorhinolaryngology, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands;
- 2Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands;
- 3Department of Medical BioSciences, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands;
- 4Department of Human Genetics, Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands;
- 5Fondazione Banca degli Occhi del Veneto, Zelarino, Venice 30174, Italy;
- 6Center for Medical Genetics, Ghent University Hospital, Ghent 9000, Belgium;
- 7Department of Biomolecular Medicine, Ghent University, Ghent 9000, Belgium;
- 8Department of Pharmaceutics, Ghent University, Ghent 9000, Belgium;
- 9Department of Internal Medicine and Radboud Center for Infectious Diseases (RCI), Radboud University Medical Center, Nijmegen 6525 GA, The Netherlands
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↵10 These authors contributed equally to this work.
Abstract
Sequencing technologies have long limited the comprehensive investigation of large transcripts associated with inherited retinal diseases (IRDs) like Usher syndrome, which involves 11 associated genes with transcripts up to 19.6 kb. To address this, we used PacBio long-read mRNA isoform sequencing (Iso-Seq) following standard library preparation and an optimized workflow to enrich for long transcripts in the human neural retina. While our workflow achieved sequencing of transcripts up to 15 kb, this was insufficient for Usher syndrome–associated genes USH2A and ADGRV1, with transcripts of 18.9 kb and 19.6 kb, respectively. To overcome this, we employed the Samplix Xdrop System for indirect target enrichment of cDNA, a technique typically used for genomic DNA capture. This method facilitated the successful capture and sequencing of ADGRV1 transcripts as well as full-length 18.9 kb USH2A transcripts. By combining algorithmic analysis with detailed manual curation of sequenced reads, we identified novel isoforms characterized by an alternative 5′ transcription start site, the inclusion of previously unannotated exons, or alternative splicing events across the 11 Usher syndrome–associated genes. These findings have significant implications for genetic diagnostics and therapeutic development. The analysis applied here on Usher syndrome–associated transcripts exemplifies a valuable approach that can be extended to explore the transcriptomic complexity of other IRD-associated genes in the complete transcriptome data set generated within this study. Additionally, we demonstrate the adaptability of the Samplix Xdrop System for capturing cDNA, and the optimized methodologies described can be expanded to facilitate the enrichment of large transcripts from various tissues of interest.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.280060.124.
- Received September 24, 2024.
- Accepted February 11, 2025.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
