title: Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk.
abstract: BACKGROUND
  Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques.
  
  
  METHODS
  Twenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA).
  
  
  RESULTS
  NGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel.
  
  
  CONCLUSIONS
  This study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. tjudkins@myriad.com.
authors.forename: Thaddeus
authors.initials: T
authors.lastname: Judkins
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. bleclair@myriad.com.
authors.forename: Benoît
authors.initials: B
authors.lastname: Leclair
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. kbowles@myriad.com.
authors.forename: Karla
authors.initials: K
authors.lastname: Bowles
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. ngutin@myriad.com.
authors.forename: Natalia
authors.initials: N
authors.lastname: Gutin
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. jtrost@myriad.com.
authors.forename: Jeff
authors.initials: J
authors.lastname: Trost
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. jmccullo@myriad.com.
authors.forename: James
authors.initials: J
authors.lastname: McCulloch
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. satishb@myriad.com.
authors.forename: Satish
authors.initials: S
authors.lastname: Bhatnagar
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. amurray@myriad.com.
authors.forename: Adam
authors.initials: A
authors.lastname: Murray
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. jcraft@myriad.com.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Craft
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. bwardell@myriad.com.
authors.forename: Bryan
authors.initials: B
authors.lastname: Wardell
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. mbastian@myriad.com.
authors.forename: Mark
authors.initials: M
authors.lastname: Bastian
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. jeff@myriad.com.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Mitchell
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. jchen@myriad.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. thanh@myriad.com.
authors.forename: Thanh
authors.initials: T
authors.lastname: Tran
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. dtrem@myriad.com.
authors.forename: Deborah
authors.initials: D
authors.lastname: Williams
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. jpotter@myriad.com.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Potter
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. sjammula@myriad.com.
authors.forename: Srikanth
authors.initials: S
authors.lastname: Jammulapati
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. mperry@myriad.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Perry
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. bmorris@myriad.com.
authors.forename: Brian
authors.initials: B
authors.lastname: Morris
authors.affiliation: Myriad Genetic Laboratories, Inc., Salt Lake City, Utah, USA. broa@myriad.com.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Roa
authors.affiliation: Myriad Genetics, Inc., Salt Lake City, Utah, USA. ktimms@myriad.com.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Timms
mesh_terms: D055028:Comparative Genomic Hybridization; D019295:Computational Biology / Q000379:methods; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D023281:Genomics / Q000379:methods; D018095:Germ-Line Mutation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D009386:Neoplastic Syndromes, Hereditary / Q000453:epidemiology* / Q000235:genetics*; D015203:Reproducibility of Results; D012306:Risk; D012680:Sensitivity and Specificity