A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing

Kelly Quek, Katia Nones, Ann-Marie Patch, J. Lynn Fink, Felicity Newell, Nicole Cloonan, David Miller, Muhammad Z. H. Fadlullah, Karin Kassahn, Angelika N. Christ, Timothy J. C. Bruxner, Suzanne Manning, Ivon Harliwong, Senel Idrisoglu, Craig Nourse, Ehsan Nourbakhsh, Shivangi Wani, Anita Steptoe, Matthew Anderson, Oliver Holmes & 10 others Conrad Leonard, Darrin Taylor, Scott Wood, Qinying Xu, Australian Pancreatic Cancer Genome Initiative, Peter Wilson, Andrew V. Biankin, John V. Pearson, Nic Waddell, Sean M. Grimmond

Research output: Contribution to journalArticle

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.
Original languageEnglish
Pages (from-to)31-38
Number of pages8
JournalBioTechniques
Volume57
Issue number1
DOIs
Publication statusPublished - 1 Jul 2014
Externally publishedYes

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Keywords

  • next-generation sequencing
  • cancer
  • chromosome breakpoints
  • structural variation
  • verification
  • high-throughput

Cite this

Quek, K., Nones, K., Patch, A-M., Fink, J. L., Newell, F., Cloonan, N., ... Grimmond, S. M. (2014). A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing. BioTechniques, 57(1), 31-38. https://doi.org/10.2144/000114189