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.
Bibliographical noteFunding Information:
The AOCS was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729, The Cancer Council Tasmania and The Cancer Foundation of Western Australia and the National Health and Medical Research Council of Australia (NHMRC, ID400413), the Peter MacCallum Cancer Foundation and Ovarian Cancer Australia (OCA). This work was supported by research grants from the Victorian Comprehensive Cancer Centre, the Victorian Cancer Agency (TRP13088), and the National Health and Medical Research Council(APP1034301) with infrastructure support for sequencing from the University of Melbourne and Therapeutic Innovation Australia. The Victorian Cancer Biobank is supported by the Victorian Government. GT is part supported by the Herman Trust. The authors thank MRC-Holland for making the probe sequences used in MLPA kits publically available. The authors thank Kathryn Alsop (AOCS), Benhur Amanuel (PathWest), Michael Christie (Royal Melbourne Hospital), Diar Aziz and Irma Gresshoff (University of Melbourne) and Audrey Partenen (Victorian Cancer Biobank) for providing DNA and tumour samples with known genotypes. The authors thank Kym Pham (University of Melbourne) for sequencing infrastructure management and support. The authors also thank Kandavel Shanmugan, Andrea Muranyi and Leanne Hendrickson (Ventana) for providing the chr19q12 ISH probe, protocol and reagents. The management group of the AOCS group included D Bowtell, G Chenevix-Trench, A Green, P Webb, A DeFazio and D Gertig. The project and data managers included N Traficante, S Fereday, S Moore, J Hung, K Harrap, T Sadkowsky, N Pandeya. The Reseach Nurses and Assistants of the AOCS group included: MMalt, A Mellon, R Robertson, T Vanden Bergh, MJones, P Mackenzie, J Maidens, K Nattress, YE Chiew, A Stenlake, H Sullivan, B Alexander, P Ashover, S Brown, T Corrish, L Green, L Jackman, K Ferguson, K Martin, A Martyn, B Ranieri, J White, V Jayde, P Mamers, L Bowes, L Galletta, D Giles, J Hendley, K Alsop, T Schmidt, H Shirley, C Ball, C Young, S Viduka, Hoa Tran, Sanela Bilic, Lydia Glavinas, Julia Brooks. The Clinical and Scientific Collaborators of the AOCS group included: R Stuart-Harris, F Kirsten, J Rutovitz, P Clingan, A Glasgow, A Proietto, S Braye, G Otton, J Shannon, T Bonaventura, J Stewart, S Begbie MFriedlander D Bell, S Baron-Hay, A Ferrier (dec.), G Gard, D Nevell, N Pavlakis, S Valmadre, B Young, C Camaris, R Crouch, L Edwards, N Hacker, D Marsden, G Robertson, P Beale, J Beith, J Carter, C Dalrymple, R Houghton, P Russell, M Links, J Grygiel, J Hill, A Brand, K Byth, R Jaworski, P Harnett, R Sharma, G Wain, B Ward, D Papadimos, A Crandon, MCummings, K Horwood, A Obermair, L Perrin, D Wyld, J Nicklin, MDavy, MK Oehler, C Hall, T Dodd, T Healy, K Pittman, D Henderson, J Miller, J Pierdes, P Blomfield, D Challis, R McIntosh, A Parker, B Brown, R Rome, D Allen, P Grant, S Hyde, R Laurie, MRobbie, D Healy, T Jobling, T Manolitsas, J McNealage, P Rogers, B Susil, E Sumithran, I Simpson, K Phillips, D Rischin, S Fox, D Johnson, S Lade, MLoughrey, N O?Callaghan, WMurray, P Waring, V Billson, J Pyman, D Neesham, MQuinn, C Underhill, R Bell, LF Ng, R Blum, V Ganju, I Hammond, Y Leung, A McCartney (dec.), MBuck, I Haviv, D Purdie, D Whiteman, N Zeps. We also acknowledge the contribution of the study nurses and research assistants and would like to thank all of the women who participated in the study.