title: Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.
abstract: BACKGROUND
  In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours.
  
  
  METHODS
  DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array.
  
  
  RESULTS
  The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton.
  
  
  CONCLUSIONS
  The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Singh
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: K P
authors.initials: KP
authors.lastname: Patel
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Routbort
authors.affiliation: Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: K
authors.initials: K
authors.lastname: Aldape
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: X
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: J
authors.initials: J
authors.lastname: Manekia
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: R
authors.initials: R
authors.lastname: Abraham
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: N G
authors.initials: NG
authors.lastname: Reddy
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Barkoh
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: J
authors.initials: J
authors.lastname: Veliyathu
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Medeiros
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: R
authors.initials: R
authors.lastname: Luthra
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics; D056915:DNA Copy Number Variations; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009154:Mutation / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D009363:Neoplasm Proteins; D004247:DNA