title: Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis.
abstract: Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.
authors.affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.
authors.forename: Laila C
authors.initials: LC
authors.lastname: Schenkel
authors.affiliation: Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kerkhof
authors.affiliation: Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada.
authors.forename: Alan
authors.initials: A
authors.lastname: Stuart
authors.affiliation: Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada.
authors.forename: Jack
authors.initials: J
authors.lastname: Reilly
authors.affiliation: Department of Pathology and Laboratory Medicine, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, Hamilton, Ontario, Canada.
authors.forename: Barry
authors.initials: B
authors.lastname: Eng
authors.affiliation: Department of Pathology and Laboratory Medicine, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, Hamilton, Ontario, Canada.
authors.forename: Crystal
authors.initials: C
authors.lastname: Woodside
authors.affiliation: Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada.
authors.forename: Alexander
authors.initials: A
authors.lastname: Levstik
authors.affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Howlett
authors.affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; Biochemical Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, London, Ontario, Canada.
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Rupar
authors.affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada.
authors.forename: Joan H M
authors.initials: JHM
authors.lastname: Knoll
authors.affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Ainsworth
authors.affiliation: Department of Pathology and Laboratory Medicine, Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, Hamilton, Ontario, Canada.
authors.forename: John S
authors.initials: JS
authors.lastname: Waye
authors.affiliation: Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada; Molecular Genetics Laboratory, Molecular Diagnostics Division, London Health Sciences Centre, Children's Health Research Institute, London, Ontario, Canada. Electronic address: bekim.sadikovic@lhsc.on.ca.
authors.forename: Bekim
authors.initials: B
authors.lastname: Sadikovic
mesh_terms: D000483:Alleles; D015331:Cohort Studies; D056915:DNA Copy Number Variations; D004272:DNA, Mitochondrial; D015723:Gene Library; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards*
chemical_list: D004272:DNA, Mitochondrial