Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing

Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here researchers from the University of British Columbia investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing.

Known or suspected deleterious germline SVs were identified using Illumina genome sequencing across a cohort of 669 advanced cancer patients with paired tumor genome and transcriptome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing.

Nanopore sequencing confirmed eight simple pathogenic or likely pathogenic SVs, resolving three additional variants whose impact could not be fully elucidated through short-read sequencing. A recurrent sequencing artifact on chromosome 16p13 and one complex rearrangement on chromosome 5q35 were subsequently classified as likely benign, obviating the need for further clinical assessment. Variant configuration was further resolved in one case with a complex pathogenic rearrangement affecting TSC2.

Schematic representations of candidate structural variants resolved using long-read sequencing

figure1

(a) A recurrent event identified in cases 1, 2, and 3 and predicted to be pathogenic was reinterpreted as a likely benign intronic variant based on Oxford nanopore sequencing. Illumina short-read genome sequencing data supported a long-range inversion on chromosome 16p13 with breakpoints in IFT140 and TSC2 (upper), while nanopore sequencing data showed an insertion in intron 30 of IFT140 likely arising from an Alu element in intron 16 of TSC2 (lower). (bc) A pathogenic complex variant in case 4 (b) and a likely benign variant in case 5 (c) characterized by nanopore sequencing. The path of long-read alignments to the reference genome is denoted by a solid black arrow, indicating the putative direction of DNA replication on the variant allele with dashed lines indicating positions of template switching and reinitiation of replication.

These findings demonstrate that long-read sequencing can improve the validation, resolution, and classification of germline SVs. This has important implications for return of results, cascade carrier testing, cancer screening, and prophylactic interventions.

Thibodeau ML, O’Neill K, Dixon K, et al. (2020) Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing. Genet Med [published online ahead of print]. [article]

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