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

My Linh Thibodeau,Sean Young,Katherine Dixon,Kieran O’Neill,David F Schaeffer,Tina Wong,Steven J.M Jones,Caralyn Reisle,Kane Tse,Stephen Yip,Richard A Moore ,Daniel J Renouf ,Howard John Lim ,Karen Mungall ,Aly Karsan,Eric Chuah,Dean Cheng,Martin Krzywinski,Erin Pleasance,Stephen Chia,Janessa Laskin,Carol Cremin,Sophie Sun,Stephen Pleasance,Yaoqing Shen,Marco A Marra,Kasmintan A Schrader,Pawan Pandoh,Alexandra Fok

doi:10.1038/s41436-020-0880-8

Abstract

PurposeStructural 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. MethodsKnown 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. ResultsNanopore 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. ConclusionOur 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.

Highlights

A significant amount of genetic variation in the human genome is due to structural variants (SVs), such as deletions, duplications, inversions, and translocations.[1,2] Genome sequencing allows high-resolution hypothesis-free analysis of variants in known and novel disease genes, and may improve rates of molecular diagnosis by overcoming some of the limitations of targeted clinical assays
Short-read sequencing Short-read genome sequencing was previously performed on Illumina HiSeq platforms in normal tissue samples for 669 advanced cancer patients enrolled in the BC Cancer Personalized OncoGenomics (POG) program (NCT02155621).[6]
Twelve candidate germline SVs were identified in 14 individuals by short-read genome sequencing, of whom 5 were known carriers (Table 1)

Summary

Introduction

A significant amount of genetic variation in the human genome is due to structural variants (SVs), such as deletions, duplications, inversions, and translocations.[1,2] Genome sequencing allows high-resolution hypothesis-free analysis of variants in known and novel disease genes, and may improve rates of molecular diagnosis by overcoming some of the limitations of targeted clinical assays. Accurate alignment and variant calling in NGS is challenging due to regions of low sequence complexity, repetitive elements, and strong GC bias in the human genome, reducing the sensitivity and specificity for novel variant discovery. This indicates a need for improved approaches to characterize genetic variation, for large or complex variants. The prevalence of SVs in clinical and research cancer cohorts is likely underestimated due to the technical and computational limitations of multigene panel sequencing, exome sequencing, and genome sequencing.[3] Recently, long-read sequencing (LRS) has been used to characterize complex genetic variation in human genomes and aid in the diagnosis of rare disorders.[4,5] To investigate the contribution of germline SVs to cancer susceptibility, we used short- and long-read genome sequencing to elucidate pathogenic

Methods

Results

Conclusion