High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

• Published in: PloS one Vol. 10; no. 11; p. e0143006
• Main Authors: Kondrashova, Olga, Love, Clare J, Lunke, Sebastian, Hsu, Arthur L, Waring, Paul M, Taylor, Graham R
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.11.2015; Public Library of Science (PLoS)
• Subjects: Amplification; BRCA1 protein; BRCA1 Protein - genetics; BRCA1 Protein - metabolism; BRCA2 protein; BRCA2 Protein - genetics; BRCA2 Protein - metabolism; Breast cancer; Care and treatment; Copy number; Councils; Cyclin E - genetics; Cyclin E - metabolism; Deoxyribonucleic acid; Development and progression; DNA; DNA Copy Number Variations - genetics; DNA probes; ErbB-2 protein; Female; Formaldehyde; Formaldehyde - chemistry; Format; Gastroesophageal reflux; Gene amplification; Gene Dosage; Gene expression; Genes, Neoplasm; Genetic aspects; High-Throughput Nucleotide Sequencing - methods; Humans; Hybridization; Limit of Detection; Medical research; Methods; Multiplex Polymerase Chain Reaction - methods; Mutation; Oncogene Proteins - genetics; Oncogene Proteins - metabolism; Oncology; Ovarian cancer; Ovarian Neoplasms - genetics; Paraffin; Paraffin Embedding; Pathology; Receptor, ErbB-2 - genetics; Receptor, ErbB-2 - metabolism; Reproducibility of Results; Sensitivity and Specificity; Tissue Fixation; Tumors; United States; Australia; Western Australia Australia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0143006

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.