Open Access Research article

Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2

Tú Nguyen-Dumont1, Zhi L Teo1, Bernard J Pope23, Fleur Hammet1, Maryam Mahmoodi1, Helen Tsimiklis1, Nelly Sabbaghian4, Marc Tischkowitz5, William D Foulkes46, Kathleen Cuningham Foundation Consortium for research into Familial Breast cancer (kConFab), Graham G Giles78, John L Hopper7, Australian Breast Cancer Family Registry7, Melissa C Southey1 and Daniel J Park1*

Author Affiliations

1 Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, VIC 3010, Australia

2 Victorian Life Sciences Computation Initiative, 187 Grattan Street, Carlton, Melbourne, VIC 3010, Australia

3 Department of Computing and Information Systems, The University of Melbourne, Melbourne, VIC 3010, Australia

4 Jewish General Hospital, Lady Davis Institute, Montréal, QC H3T 1E2, Canada

5 Department of Medical Genetics, University of Cambridge, Cambridge, UK

6 Departments of Oncology and Human Genetics, McGill University, Montréal, QC H2W 1S6, Canada

7 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, The University of Melbourne, Melbourne, VIC 3010, Australia

8 Cancer Epidemiology Centre, The Cancer Council Victoria, Rathdowne Street, Carlton 3052, Australia

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BMC Medical Genomics 2013, 6:48  doi:10.1186/1755-8794-6-48

Published: 8 November 2013



Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour.


Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run.


We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing.


These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.

Hi-Plex; Massively parallel sequencing; Mutation screening; PALB2; Molecular diagnostics