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

ABSTRACT

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
• retinoblastoma
• long-read sequencing
• adaptive sampling
• targeted sequencing
• haplotyping
• methylation

Acknowledgements

The authors would like to thank Angela Sandt in the Van Gelder lab for technical assistance. We acknowledge the Northwest Genomics Center for providing assistance for Illumina library preparation and performing short-read genome sequencing on the Illumina NovaSeq6000 platform. We thank Dr. Danny Miller and his laboratory at the University of Washington for library preparation and performing long-read genome sequencing on the ONT PromethION platform.

Disclosure statement

The authors report no conflicts of interest. The authore alone are responsible for the content and writring of this article.

Additional information

Funding

This work was supported by an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology at University of Washington; The Sinksey Foundation (DM); and the Violet Sees Foundation (AWS, DM).