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

Abstract

Purpose Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here we investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing. Methods 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. Results 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. Conclusion Our 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.

How Should We Test for Lynch Syndrome? A Review of Current Guidelines and Future Strategies

“…For example, short reads are generally considered to have a poorer sensitivity for detecting structural variants than long reads [ 133 ]. Recently, long-read sequencing was used to clarify suspected structural variants in the germlines of hereditary cancer patients [ 134 ], and there are known examples of large deletions or rearrangements causing LS, such as the Finnish MLH1 founder variant [ 85 , 92 ] and Australian MHS2 inversion [ 93 ] described earlier. In addition, novel base-callers being developed for long-read sequencing data may be able to detect base modifications from subtle effects on sequencing dynamics [ 132 ], which could facilitate detection of germline epigenetic modifications of MMR genes, such as those estimated to account for 2–3% of MLH1 -associated LS [ 73 , 74 , 75 ].…”

Intronic Breakpoint Signatures Enhance Detection and Characterization of Clinically Relevant Germline Structural Variants

“…The first rearrangement, which does not result in any copy number change of coding exons, introduces a singleexon inversion. Thibodeau et al 8 recently reported this same inversion, and RNA sequencing demonstrated that this results in skipping of exon 5. The second rearrangement constitutes a large deletion with six insertion components, including a 2.7kb sequence in inverted orientation.…”

“…In addition, long-read sequencing simplifies the characterization of breakpoint signatures and is helpful to resolve suspicious or complex signals detected in short-read sequencing. 8 The SVs discovered in germline testing exhibit many features that are also found in somatic SVs. 65 Thus, many SV detection algorithms show equivalent performance for germline sequencing and tumor testing with variant allele fractions as low as 20%.…”

A Distributed Whole Genome Sequencing Benchmark Study

“…It is also important to consider the source of the DNA sample (Trost et al, 2019), which can influence the quality and amount of input DNA used for sequencing. Each of these factors may in turn impact all aspects of data generated, in particular when long-read technologies are used rather than the short-read sequencing presented here (Wang et al, 2019;Thibodeau et al, 2020).…”