title: Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2.
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.
authors.forename: Tú
authors.initials: T
authors.lastname: Nguyen-Dumont
authors.forename: Zhi L
authors.initials: ZL
authors.lastname: Teo
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Pope
authors.forename: Fleur
authors.initials: F
authors.lastname: Hammet
authors.forename: Maryam
authors.initials: M
authors.lastname: Mahmoodi
authors.forename: Helen
authors.initials: H
authors.lastname: Tsimiklis
authors.forename: Nelly
authors.initials: N
authors.lastname: Sabbaghian
authors.forename: Marc
authors.initials: M
authors.lastname: Tischkowitz
authors.forename: William D
authors.initials: WD
authors.lastname: Foulkes
authors.forename: Graham G
authors.initials: GG
authors.lastname: Giles
authors.forename: John L
authors.initials: JL
authors.lastname: Hopper
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Southey
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, VIC 3010, Australia. djp@unimelb.edu.au.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Park
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D000076164:Fanconi Anemia Complementation Group N Protein; D005260:Female; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D000076164:Fanconi Anemia Complementation Group N Protein; D009687:Nuclear Proteins; C512790:PALB2 protein, human; D025521:Tumor Suppressor Proteins