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, Australian Pancreatic Cancer Genome Initiative, Wilson, Peter, Biankin, Andrew V., Pearson, John V., Waddell, Nic and Grimmond, Sean M. (2014) A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing. BioTechniques, 57 1: 31-38. doi:10.2144/000114189

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Author 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
Australian Pancreatic Cancer Genome Initiative
Wilson, Peter
Biankin, Andrew V.
Pearson, John V.
Waddell, Nic
Grimmond, Sean M.
Title A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing
Journal name BioTechniques   Check publisher's open access policy
ISSN 1940-9818
Publication date 2014-07-01
Year available 2014
Sub-type Article (original research)
DOI 10.2144/000114189
Volume 57
Issue 1
Start page 31
End page 38
Total pages 8
Place of publication New York, United States
Publisher Informa Healthcare
Language eng
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.
Keyword Cancer
Chromosome breakpoints
Next-generation sequencing
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2015 Collection
Institute for Molecular Bioscience - Publications
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