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

Tú Nguyen-Dumont, Zhi Ling Teo, Bernard J. Pope, Fleur Hammet, Maryam Mahmoodi, Helen Tsimiklis, Nelly Sabbaghian, Marc Tischkowitz, William D Foulkes, Graham G. Giles, John L. Hopper, Australian Breast Cancer Family Registry, Melissa C. Southey, Daniel J. Park

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13 Citations (Scopus)


Background: 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. Methods. 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. Results: 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. Conclusions: 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.

Original languageEnglish
Article number48
Number of pages5
JournalBMC Medical Genomics
Issue number1
Publication statusPublished - 2013
Externally publishedYes


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

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