title: A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing.
abstract: Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Kelly
authors.initials: K
authors.lastname: Quek
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Katia
authors.initials: K
authors.lastname: Nones
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Ann-Marie
authors.initials: AM
authors.lastname: Patch
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: J Lynn
authors.initials: JL
authors.lastname: Fink
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Felicity
authors.initials: F
authors.lastname: Newell
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Nicole
authors.initials: N
authors.lastname: Cloonan
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Miller
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Muhammad Z H
authors.initials: MZ
authors.lastname: Fadlullah
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Karin
authors.initials: K
authors.lastname: Kassahn
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Angelika N
authors.initials: AN
authors.lastname: Christ
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Timothy J C
authors.initials: TJ
authors.lastname: Bruxner
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Manning
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Ivon
authors.initials: I
authors.lastname: Harliwong
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Senel
authors.initials: S
authors.lastname: Idrisoglu
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Craig
authors.initials: C
authors.lastname: Nourse
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Nourbakhsh
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Shivangi
authors.initials: S
authors.lastname: Wani
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Anita
authors.initials: A
authors.lastname: Steptoe
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Matthew
authors.initials: M
authors.lastname: Anderson
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Oliver
authors.initials: O
authors.lastname: Holmes
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Conrad
authors.initials: C
authors.lastname: Leonard
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Darrin
authors.initials: D
authors.lastname: Taylor
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Scott
authors.initials: S
authors.lastname: Wood
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Qinying
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Wilson
authors.affiliation: The Kinghorn Cancer Centre, Cancer Research Program, Garvan Institute of Medical Research, Sydney, NSW, Australia; Department of Surgery, Bankstown Hospital, Sydney, NSW, Australia; South Western Sydney Clinical School, Faculty of Medicine, University of NSW, Liverpool, NSW, Australia; Wolfson Wohl Cancer Research Centre, Institute for Cancer Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Biankin
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: John V
authors.initials: JV
authors.lastname: Pearson
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
authors.forename: Nic
authors.initials: N
authors.lastname: Waddell
authors.affiliation: Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia; Wolfson Wohl Cancer Research Centre, Institute for Cancer Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Grimmond
mesh_terms: D001483:Base Sequence; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics; D002869:Chromosome Aberrations; D056905:Chromosome Breakpoints; D002877:Chromosomes, Human; D017931:DNA Primers; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D057188:Workflow
chemical_list: D017931:DNA Primers
keywords: cancer; chromosome breakpoints; high-throughput; next-generation sequencing; structural variation; verification