Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk
| Autor: | Mark Bastian, Jian Chen, James McCulloch, Thanh Tran, J.T. Mitchell, Karla R. Bowles, Natalia Gutin, Srikanth Jammulapati, Deborah Williams, Jeffrey T. Trost, Bryan Wardell, Kirsten Timms, Adam Murray, Jonathan Craft, Jennifer Potter, Benoît Leclair, Michael C. Perry, Benjamin B. Roa, Brian Morris, Thaddeus Judkins, Satish Bhatnagar |
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| Rok vydání: | 2015 |
| Předmět: |
Risk
Cancer Research Genes BRCA2 Genes BRCA1 Genomics Biology Sensitivity and Specificity DNA sequencing symbols.namesake Germline mutation Neoplastic Syndromes Hereditary Next generation sequencing Genetics medicine Humans Genetic Predisposition to Disease Multiplex Genetic Testing Multiplex ligation-dependent probe amplification Germ-Line Mutation Genetic testing Sanger sequencing Comparative Genomic Hybridization medicine.diagnostic_test Computational Biology High-Throughput Nucleotide Sequencing Reproducibility of Results BRCA1 BRCA2 Hereditary cancer Technical Advance Oncology Mutation symbols Comparative genomic hybridization |
| Zdroj: | BMC Cancer |
| ISSN: | 1471-2407 |
| DOI: | 10.1186/s12885-015-1224-y |
| Popis: | 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. Conclusion 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. |
| Databáze: | OpenAIRE |
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