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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.

Type Journal
ISBN 1940-9818 (Electronic) 0736-6205 (Linking)
Authors Quek, K.; Nones, K.; Patch, A. M.; Fink, J. L.; Newell, F.; Cloonan, N.; Miller, D.; Fadlullah, M. Z.; Kassahn, K.; Christ, A. N.; Bruxner, T. J.; Manning, S.; Harliwong, I.; Idrisoglu, S.; Nourse, C.; Nourbakhsh, E.; Wani, S.; Steptoe, A.; Anderson, M.; Holmes, O.; Leonard, C.; Taylor, D.; Wood, S.; Xu, Q.; Australian Pancreatic Cancer Genome, Initia
Garvan Authors Joanne Scorer
Publisher Name BIOTECHNIQUES
Published Date 2014-01-01 00:00:00
Published Volume 57
Published Issue 1
Published Pages 31-8
URL http://www.ncbi.nlm.nih.gov/pubmed/25005691
Status Published In-print
OpenAccess Link https://publications.gimr.garvan.org.au/download.php?12424_12777/BTN_A_000114189_O_244524a(1).pdf