Deciphering the largest disease-associated transcript isoforms in the human neural retina with advanced long-read...

Area of Science:

• Genomics and Transcriptomics
• Ophthalmology and Vision Science
• Molecular Biology

Background:

• Sequencing technologies have historically struggled to comprehensively analyze large transcripts, particularly those implicated in inherited retinal diseases (IRDs).
• Usher syndrome, a major cause of IRDs, involves 11 genes with transcripts up to 19.6 kb, posing significant challenges for full-length sequencing.
• Existing PacBio long-read mRNA isoform sequencing (Iso-Seq) workflows with standard enrichment were insufficient for capturing the longest Usher syndrome transcripts.

Purpose of the Study:

• To develop and optimize a method for enriching and sequencing long transcripts associated with Usher syndrome and other IRDs.
• To identify novel transcript isoforms and alternative splicing events in Usher syndrome-associated genes.
• To demonstrate the utility of the Samplix Xdrop System for capturing long cDNA targets.

Main Methods:

• Utilized PacBio long-read Iso-Seq on human neural retina samples.
• Implemented an optimized workflow for long transcript enrichment.
• Employed the Samplix Xdrop System for indirect target enrichment of cDNA to capture previously inaccessible long transcripts (e.g., USH2A, ADGRV1).

Main Results:

• Successfully captured and sequenced full-length transcripts for Usher syndrome-associated genes, including 18.9 kb USH2A and 19.6 kb ADGRV1.
• Identified novel transcript isoforms through algorithmic analysis and manual curation, revealing alternative 5' start sites, unannotated exons, and alternative splicing events.
• Demonstrated the Samplix Xdrop System's effectiveness for cDNA enrichment, extending its application beyond genomic DNA.

Conclusions:

• The optimized workflow and Samplix Xdrop System enable comprehensive transcriptomic analysis of large genes in IRDs like Usher syndrome.
• Discovery of novel isoforms provides critical insights for advancing genetic diagnostics and developing targeted therapies for inherited retinal diseases.
• The described methodologies are adaptable for enriching large transcripts from diverse tissues, facilitating broader transcriptomic research.