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Updated: Jan 10, 2026
Use of Alu Element Containing Minigenes to Analyze Circular RNAs
Published on: March 10, 2020
Limitations of short-read NGS in detecting RP1 Alu insertions: a case emphasizing Sanger confirmation
Takaaki Hayashi1, Kei Mizobuchi1, Natsuki Higa1
1Department of Ophthalmology, The Jikei University School of Medicine, Tokyo, Japan.
Purpose:
To assess the detectability of the pathogenic RP1 Alu insertion using the PrismGuide™ inherited retinal dystrophy (IRD) panel, which targets 82 IRD genes, and whole-exome sequencing (WES).
Methods:
A girl diagnosed with early-onset retinitis pigmentosa at age 7 underwent IRD panel testing and WES at age 15. Sequencing data from both platforms were evaluated using standard automated pipelines and manually reviewed with the Integrative Genomics Viewer (IGV). PCR and Sanger sequencing were performed for confirmation.
Results:
Automated pipelines for both the IRD panel and WES failed to detect any reportable pathogenic variants. However, IGV review revealed a markedly reduced read coverage within exon 4 of RP1 in both datasets, suggesting the homozygous Alu insertion. PCR and Sanger sequencing confirmed the presence of the insertion. Thus, both short-read sequencing approaches failed to identify the variant.
Conclusions:
Short-read sequencing technologies, including the IRD panel and WES, have limitations in detecting short interspersed nuclear elements such as the RP1 Alu insertion. This case emphasizes the importance of manual IGV review and the necessity of confirmatory Sanger sequencing when such structural variants are suspected despite negative automated analyses.
