Hi-Plex for High-Throughput Mutation Screening: Application to the Breast Cancer Susceptibility Gene PALB2

BMC Med Genomics. 2013 Nov 8;6:48. doi: 10.1186/1755-8794-6-48.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / genetics*
  • DNA Mutational Analysis / methods*
  • Fanconi Anemia Complementation Group N Protein
  • Female
  • Genetic Predisposition to Disease / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Mutation
  • Nuclear Proteins / genetics*
  • Tumor Suppressor Proteins / genetics*

Substances

  • Fanconi Anemia Complementation Group N Protein
  • Nuclear Proteins
  • PALB2 protein, human
  • Tumor Suppressor Proteins