title: Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours.
abstract: Massively parallel sequencing offers the ability to interrogate a tumour biopsy for multiple mutational changes. For clinical samples, methodologies must enable maximal extraction of available sequence information from formalin-fixed and paraffin-embedded (FFPE) material. We assessed the use of targeted capture for mutation detection in FFPE DNA. The capture probes targeted the coding region of all known kinase genes and selected oncogenes and tumour suppressor genes. Seven melanoma cell lines and matching FFPE xenograft DNAs were sequenced. An informatics pipeline was developed to identify variants and contaminating mouse reads. Concordance of 100% was observed between unfixed and formalin-fixed for reported COSMIC variants including BRAF V600E. mutations in genes not conventionally screened including ERBB4, ATM, STK11 and CDKN2A were readily detected. All regions were adequately covered with independent reads regardless of GC content. This study indicates that hybridisation capture is a robust approach for massively parallel sequencing of FFPE samples.
authors: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia.
authors: Stephen Q
authors: SQ
authors: Wong
authors: Bioinformatics Core, Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.
authors: Jason
authors: J
authors: Li
authors: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia.
authors: Renato
authors: R
authors: Salemi
authors: 1] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia [2] Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia [3] Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia.
authors: Karen E
authors: KE
authors: Sheppard
authors: 1] Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia [2] Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, The Olivia Newton-John Cancer and Wellness Centre, Heidelberg, Victoria, 3084, Australia.
authors: Hongdo
authors: H
authors: Do
authors: 1] Bioinformatics Core, Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia [2] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia [3] Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia.
authors: Richard W
authors: RW
authors: Tothill
authors: 1] Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia [2] Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia [3] Division of Cancer Research, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, 3002, Australia.
authors: Grant A
authors: GA
authors: McArthur
authors: 1] Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia [2] Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia [3] Translational Genomics and Epigenomics Laboratory, Ludwig Institute for Cancer Research, The Olivia Newton-John Cancer and Wellness Centre, Heidelberg, Victoria, 3084, Australia.
authors: Alexander
authors: A
authors: Dobrovic
mesh_terms: D000818:Animals; D016477:Artifacts; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D005091:Exons; D020862:GC Rich Sequence; D064593:Heterografts; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054643:INDEL Mutation; D051379:Mice; D009154:Mutation; D059645:Mutation Rate; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D020641:Polymorphism, Single Nucleotide; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D048493:Proto-Oncogene Proteins B-raf