title: High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.
abstract: Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.
authors.affiliation: Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Olga
authors.initials: O
authors.lastname: Kondrashova
authors.affiliation: Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Clare J
authors.initials: CJ
authors.lastname: Love
authors.affiliation: Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lunke
authors.affiliation: Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Hsu
authors.affiliation: Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Waring
authors.affiliation: Department of Pathology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Graham R
authors.initials: GR
authors.lastname: Taylor
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism; D024682:BRCA2 Protein / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D056915:DNA Copy Number Variations / Q000235:genetics; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D018628:Gene Dosage; D052138:Genes, Neoplasm; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein; D019927:Cyclin E; D005557:Formaldehyde; D015513:Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; C475820:CCNE1 protein, human; C508053:ERBB2 protein, human