Your browser doesn't support javascript.
loading
A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing.
Quek, Kelly; Nones, Katia; Patch, Ann-Marie; Fink, J Lynn; Newell, Felicity; Cloonan, Nicole; Miller, David; Fadlullah, Muhammad Z H; Kassahn, Karin; Christ, Angelika N; Bruxner, Timothy J C; Manning, Suzanne; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Wani, Shivangi; Steptoe, Anita; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Wilson, Peter; Biankin, Andrew V; Pearson, John V; Waddell, Nic; Grimmond, Sean M.
Affiliation
  • Quek K; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Nones K; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Patch AM; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Fink JL; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Newell F; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Cloonan N; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Miller D; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Fadlullah MZ; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Kassahn K; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Christ AN; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Bruxner TJ; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Manning S; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Harliwong I; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Idrisoglu S; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Nourse C; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Nourbakhsh E; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Wani S; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Steptoe A; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Anderson M; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Holmes O; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Leonard C; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Taylor D; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Wood S; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Xu Q; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Wilson P; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Biankin AV; 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
  • Pearson JV; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Waddell N; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD, Australia.
  • Grimmond SM; 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.
Biotechniques ; 57(1): 31-8, 2014 Jul.
Article in En | MEDLINE | ID: mdl-25005691
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.
Subject(s)
Key words

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Chromosomes, Human / Chromosome Breakpoints / High-Throughput Nucleotide Sequencing Limits: Humans Language: En Journal: Biotechniques Year: 2014 Document type: Article Affiliation country: Australia

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Chromosomes, Human / Chromosome Breakpoints / High-Throughput Nucleotide Sequencing Limits: Humans Language: En Journal: Biotechniques Year: 2014 Document type: Article Affiliation country: Australia