Long‐read nanopore DNA sequencing can resolve complex intragenic duplication/deletion variants, providing information to enable preimplantation genetic diagnosis

• Published in: Prenatal diagnosis Vol. 42; no. 2; pp. 226 - 232
• Main Authors: Watson, Christopher M., Holliday, Deborah L., Crinnion, Laura A., Bonthron, David T.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.02.2022; John Wiley and Sons Inc
• Subjects: Amplification; Cancer; Deletion; Deoxyribonucleic acid; Diagnosis; Diagnostic systems; DNA; DNA sequencing; Female; Gene Deletion; Gene Duplication; Genetic Markers; Genetic screening; Genetic Testing - methods; Genomic analysis; Hereditary diseases; Humans; Infant; Insertion; Male; Medical diagnosis; Multiplexing; Mutation; Nanopore Sequencing - methods; Nucleotides; Original; Polymerase chain reaction; Pregnancy; Preimplantation Diagnosis - methods; Reproduction (copying); Retina; Retinoblastoma; Retinoblastoma - diagnosis; Retinoblastoma - genetics; Retinoblastoma Binding Proteins - genetics; Sequence Analysis, DNA - methods; Technology; Ubiquitin-Protein Ligases - genetics; Whole genome sequencing
• ISSN: 0197-3851; 1097-0223; 1097-0223
• DOI: 10.1002/pd.6089

Background The adoption of massively parallel short‐read DNA sequencing methods has greatly expanded the scope and availability of genetic testing for inherited diseases. Indeed, the power of these methods has encouraged the integration of whole genome sequencing, the most comprehensive single approach to genomic analysis, into clinical practice. Despite these advances, diagnostic techniques that incompletely resolve the precise molecular boundaries of pathogenic sequence variants continue to be routinely deployed. This can present a barrier for certain prenatal diagnostic approaches. For example, the pre‐referral workup for couples seeking preimplantation genetic diagnosis requires intragenic dosage variants to be characterised at nucleotide resolution. Objective We sought to assess the use of long‐read nanopore sequencing to rapidly characterise an apparent heterozygous RB1 exon 23 deletion that was initially identified by multiplex ligation‐dependent probe amplification (MLPA), in a patient with bilateral retinoblastoma. Methods Target enrichment was performed by long‐range polymerase chain reaction (PCR) amplification prior to Flongle sequencing on a MinION long‐read sequencer. Results Characterisation of the deletion breakpoint included an unexpected 85‐bp insertion which duplicated RB1 exon 24 (and was undetected by MLPA). The long‐read sequence permitted design of a multiplex PCR assay, which confirmed that the mutation arose de novo. Conclusion Our experience demonstrates the diagnostic utility of long‐read technology for the precise characterisation of structural variants, and highlights how this technology can be efficiently deployed to enable onward referral to reproductive medicine services. Key points What's already known about this topic? Molecular diagnostic techniques that incompletely resolve pathogenic sequence variants can present a barrier for certain prenatal diagnostic approaches. What does this study add? This study demonstrates how nanopore‐based sequencing could be rapidly deployed for follow‐up analysis of previously identified, but incompletely‐defined structural variants, enabling onward referral to a national preimplantation genetic diagnosis service.