Targeted long-read sequencing allows for rapid identification of pathogenic disease-causing variants in retinoblastoma.

Publisher: Informa Healthcare Country of Publication: England NLM ID: 9436057 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1744-5094 (Electronic) Linking ISSN: 13816810 NLM ISO Abbreviation: Ophthalmic Genet Subsets: MEDLINE

Publication: London : Informa Healthcare
Original Publication: Buren, The Netherlands : Aeolus Press, c1994-

Retinoblastoma*/diagnosis ; Retinoblastoma*/genetics ; Retinoblastoma*/pathology ; Retinal Neoplasms*/pathology; Humans ; Genes, Retinoblastoma/genetics ; Sequence Analysis, DNA ; High-Throughput Nucleotide Sequencing

Background: Identification of disease-causing variants of the retinoblastoma gene (RB1), the predominant cause of retinoblastoma, is challenging. Targeted long-read genome sequencing offers a novel approach to resolve the diverse range of pathogenic variants in RB1 and provides haplotype information rapidly.
Materials and Methods: Genomic DNA was isolated from a venipuncture blood draw of a retinoblastoma patient. Whole genome sequencing (WGS) was carried out using the short-read Ilumina platform. WGS and targeted sequencing of RB1 was accomplished using the long-read Oxford Nanopore Technologies (ONT) platform. Deep-learning frameworks allowed haplotagging, variant calling, and variant annotation of both short- and long-read data.
Results: Targeted long-read sequencing of the RB1 gene allowed for enhanced depth of read coverage for discovery of rare variants and haplotype analysis. A duplication leading to a frameshift and early termination in RB1 was identified as the most deleterious variant by all sequencing methods, with long-read technology providing additional information of methylation signal and haplotype information. More importantly, there was greater than 98% concordance of RB1 variants identified between short-read and targeted long-read sequencing modalities.
Conclusions: Targeted long-read technology allows for focused sequencing effort for variant discovery. Application of this for the first time in a retinoblastoma patient allowed haplotagged variant identification and demonstrated excellent concordance with benchmark short-read sequencing. The added benefit of targeted long-read sequencing to resolve disease-causing genomic variation in RB1 rapidly from a blood draw will provide a more definitive diagnosis of heritable RB and guide management decisions for patients and their families.

Keywords: RB1; adaptive sampling; haplotyping; long-read sequencing; methylation; retinoblastoma; targeted sequencing

Date Created: 20221103 Date Completed: 20230126 Latest Revision: 20230215

20231215

10.1080/13816810.2022.2141797

36325802