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Clin Chem. 2017 Feb;63(2):503-512. doi: 10.1373/clinchem.2016.263897. Epub 2016 Dec 14.

BRCA Testing by Single-Molecule Molecular Inversion Probes.

Author information

1
Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands.
2
Department of Mechanical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands.
3
Department of Pathology, Radboud university medical center, Nijmegen, the Netherlands.
4
Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway.
5
Western Norway Familial Cancer Center, Haukeland University Hospital, Bergen, Norway.
6
Department of Clinical Science, University of Bergen, Bergen, Norway.
7
Department of Genome Sciences, University of Washington, Seattle, WA.
8
Department of Human Genetics, Radboud university medical center, Nijmegen, the Netherlands; marcel.nelen@radboudumc.nl.
9
Donders Centre for Neuroscience, Radboud University Nijmegen, Nijmegen, the Netherlands.

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.

PMID:
27974384
DOI:
10.1373/clinchem.2016.263897
[Indexed for MEDLINE]
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