BRCA Testing by Single-Molecule Molecular Inversion Probes
| Autor: | Alwin Rikken, Bart van Lier, Dimitra Zafeiropoulou, Alexander Hoischen, Ivo B J F Adan, Ronny Derks, Marcel R. Nelen, Stijn M M Bertens, Kornelia Neveling, Djie Tjwan Thung, Marloes Steehouwer, Michael Kwint, Jay Shendure, Hicham Ouchene, Evan A. Boyle, Arjen R. Mensenkamp, Jayne Y. Hehir-Kwa, Stefan H. Lelieveld, Tomasz Stokowy, Steven Castelein, Tom Hofste, Per M. Knappskog, Astrid Eijkelenboom, Beth Martin, Ermanno A.J. Bosgoed, Vidar M. Steen, Helger G. Yntema, Marjolijn J. L. Ligtenberg, Bastiaan B J Tops, Hildegunn Høberg-Vetti, Marloes Tychon |
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| Přispěvatelé: | Mechanical Engineering, Operations Planning Acc. & Control |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
DNA Copy Number Variations Clinical Biochemistry lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] Computational biology Rare cancers Radboud Institute for Molecular Life Sciences [Radboudumc 9] Biology SDG 3 – Goede gezondheid en welzijn Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] DNA sequencing 03 medical and health sciences SDG 3 - Good Health and Well-being Tumours of the digestive tract Radboud Institute for Molecular Life Sciences [Radboudumc 14] medicine False positive paradox Humans Multiplex Copy-number variation Gene CHEK2 Genetic testing BRCA2 Protein Genetics Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7] medicine.diagnostic_test BRCA1 Protein Other Research Radboud Institute for Health Sciences [Radboudumc 0] Biochemistry (medical) High-Throughput Nucleotide Sequencing genomic DNA 030104 developmental biology Neurodevelopmental disorders Radboud Institute for Molecular Life Sciences [Radboudumc 7] DNA Probes |
| Zdroj: | Clinical Chemistry, 63, 503-512 Clinical Chemistry, 63(2), 503-512. American Association for Clinical Chemistry Inc. Clinical Chemistry, 63, 2, pp. 503-512 |
| ISSN: | 0009-9147 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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