title: BRCA Testing by Single-Molecule Molecular Inversion Probes.
abstract: BACKGROUND
  Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs.
  
  
  METHODS
  The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant.
  
  
  RESULTS
  Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (>3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10-15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%.
  
  
  CONCLUSIONS
  smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Kornelia
authors.initials: K
authors.lastname: Neveling
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Arjen R
authors.initials: AR
authors.lastname: Mensenkamp
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Ronny
authors.initials: R
authors.lastname: Derks
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Michael
authors.initials: M
authors.lastname: Kwint
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Hicham
authors.initials: H
authors.lastname: Ouchene
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Marloes
authors.initials: M
authors.lastname: Steehouwer
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: van Lier
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Ermanno
authors.initials: E
authors.lastname: Bosgoed
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Alwin
authors.initials: A
authors.lastname: Rikken
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Marloes
authors.initials: M
authors.lastname: Tychon
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Zafeiropoulou
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: Castelein
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Jayne
authors.initials: J
authors.lastname: Hehir-Kwa
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Djie
authors.initials: D
authors.lastname: Tjwan Thung
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Tom
authors.initials: T
authors.lastname: Hofste
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Lelieveld
authors.affiliation: Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands.
authors.forename: Stijn M M
authors.initials: SM
authors.lastname: Bertens
authors.affiliation: Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands.
authors.forename: Ivo B J F
authors.initials: IB
authors.lastname: Adan
authors.affiliation: Department of Pathology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Astrid
authors.initials: A
authors.lastname: Eijkelenboom
authors.affiliation: Department of Pathology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Bastiaan B
authors.initials: BB
authors.lastname: Tops
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Helger
authors.initials: H
authors.lastname: Yntema
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.|Western Norway Familial Cancer Center, Haukeland University Hospital, Bergen, Norway.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Stokowy
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.
authors.forename: Per M
authors.initials: PM
authors.lastname: Knappskog
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.|Western Norway Familial Cancer Center, Haukeland University Hospital, Bergen, Norway.
authors.forename: Hildegunn
authors.initials: H
authors.lastname: Høberg-Vetti
authors.affiliation: Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.|Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Vidar M
authors.initials: VM
authors.lastname: Steen
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA.
authors.forename: Evan
authors.initials: E
authors.lastname: Boyle
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA.
authors.forename: Beth
authors.initials: B
authors.lastname: Martin
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.|Department of Pathology, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Marjolijn J L
authors.initials: MJ
authors.lastname: Ligtenberg
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA.
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands; marcel.nelen@radboudumc.nl.
authors.forename: Marcel R
authors.initials: MR
authors.lastname: Nelen
authors.affiliation: Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.|Donders Centre for Neuroscience, Radboud University Nijmegen, Nijmegen, the Netherlands.
authors.forename: Alexander
authors.initials: A
authors.lastname: Hoischen
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D056915:DNA Copy Number Variations / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D015342:DNA Probes