Gene Fusion Identification Using Anchor-Based Multiplex PCR and Next-Generation Sequencing
| Autor: | Elizabeth M Azzato, Daniel H. Farkas, Anders Meyer, Jay E. Brock, Brian P. Rubin, Maureen A. Jakubowski, Yu-Wei Cheng, Sean O Keenan, Michael D. Weindel |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
medicine.diagnostic_test High-Throughput Nucleotide Sequencing RNA General Medicine Computational biology Biology Immunohistochemistry DNA sequencing Primer extension Fusion gene 03 medical and health sciences 030104 developmental biology 0302 clinical medicine 030220 oncology & carcinogenesis Multiplex polymerase chain reaction Nucleic acid medicine Humans Gene Fusion Primer (molecular biology) Multiplex Polymerase Chain Reaction In Situ Hybridization Fluorescence Fluorescence in situ hybridization |
| Zdroj: | The Journal of Applied Laboratory Medicine. 6:917-930 |
| ISSN: | 2475-7241 2576-9456 |
| Popis: | Background Methods for identifying gene fusion events, such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and transcriptome analysis, are either single gene approaches or require bioinformatics expertise not generally available in clinical laboratories. We analytically validated a customized next-generation sequencing (NGS) panel targeting fusion events in 34 genes involving soft-tissue sarcomas. Methods Specimens included 87 formalin-fixed paraffin-embedded (FFPE) tissues with known gene fusion status. Isolated total nucleic acid was used to identify fusion events at the RNA level. The potential fusions were targeted by gene-specific primers, followed by primer extension and nested PCR to enrich for fusion candidates with subsequent bioinformatics analysis. Results The study generated results using the following quality metrics for fusion detection: (a) ≥100 ng total nucleic acid, (b) RNA average unique start sites per gene-specific primer control ≥10, (c) quantitative PCR assessing input RNA quality had a crossing point Conclusions The test validation study demonstrated analytical sensitivity of 98.7% and analytical specificity of 90.0%. The NGS-based panel generated highly concordant results compared to alternative testing methods. |
| Databáze: | OpenAIRE |
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