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

Webb, P.; Bowtell, D.; Chenevix-Trench, G.; Green, A.; Kondrashova, Olga; DeFazio, A.; Gertig, D.; Traficante, N.; Fereday, S.; Moore, S.; Hung, J.; Harrap, K.; Love, Clare J.; Sadkowsky, T.; Pandeya, N.; Malt, M.; White, J.; Mellon, A.; Robertson, R.; Vanden Bergh, T.; Jones, M.; Mackenzie, P.; Lunke, Sebastian; Maidens, J.; Young, C.; Nattress, K.; Chiew, Y. E.; Stenlake, A.; Sullivan, H.; Alexander, B.; Hsu, Arthur L.; Ashover, P.; Brown, S.; Corrish, T.; Green, L.; Jackman, L.; Ferguson, K.; Martin, K.; Martyn, A.; Young, B.; Ranieri, B.; Jayde,; Mamers, P.; Bowes, L.; Galletta, L.; Giles, D.; Hendley, J.; Alsop, K.; Schmidt, T.; Shirley, H.; Ball, C.; Viduka, S.; Tran, H.; Bilic, S.; Wyld, D.; Glavinas, L.; Brooks, J.; Stuart-Harris, R.; Kirsten, F.; Rutovitz, J.; Clingan, P.; Glasgow, A.; Proietto, A.; Braye, S.; Otton, G.; Shannon, J.; Bonaventura, T.; Stewart, J.; Begbie, S.; Friedlander, M.; Bell, D.; Baron-Hay, S.; Ferrier, A.; Gard, G.; Nevell, D.; Waring, P. M.; Pavlakis, N.; Valmadre, S.; Camaris, C.; Crouch, R.; Edwards, L.; Hacker, N.; Whiteman, D.; Marsden, D.; Zeps, N.; Robertson, G.; Beale, P.; Beith, J.; Carter, J.; Dalrymple, C.; Houghton, R.; Russell, P.; Links, M.; Grygiel, J.; Hill, J.; Brand, A.; Byth, K.; Jaworski, R.; Harnett, P.; Sharma, R.; Wain, G.; Ward, B.; Papadimos, D.; Crandon, A.; Cummings, M.; Horwood, K.; Obermair, A.; Perrin, L.; Nicklin, J.; Davy, M.; Oehler, M. K.; Hall, C.; Dodd, T.; Healy, T.; Pittman, K.; Henderson, D.; Miller, J.; Pierdes, J.; Blomfield, P.; Challis, D.; McIntosh, R.; Parker, A.; Brown, B.; Rome, R.; Allen, D.; Grant, P.; Hyde, S.; Laurie, R.; Robbie, M.; Healy, D.; Jobling, T.; Manolitsas, T.; McNealage, J.; Rogers, P.; Susil, B.; Sumithran, E.; Simpson, I.; Phillips, K.; Rischin, D.; Fox, S.; Johnson, D.; Lade, S.; Loughrey, M.; O'Callaghan, N.; Murray, W.; Billson,; Pyman, J.; Neesham, D.; Quinn, M.; Underhill, C.; Bell, R.; Ng, L. F.; Waring, Paul M.; Blum, R.; Ganju,; Hammond, I.; Leung, Y.; McCartney, A.; Buck, M.; Haviv, I.; Purdie, D.; Taylor, Graham R.

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Public Library of Science, PLoS ONE, 11(10), p. e0143006, 2015

DOI: 10.1371/journal.pone.0143006

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

Journal article published in 2015 by P. Webb, D. Bowtell, G. Chenevix-Trench, A. Green, Olga Kondrashova, A. DeFazio, D. Gertig, N. Traficante, S. Fereday, S. Moore, J. Hung, K. Harrap, Clare J. Love, T. Sadkowsky, N. Pandeya and other authors.

This paper is made freely available by the publisher.

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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.