Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis
Background: Epigenetic biomarkers, particularly CpG methylation, are increasingly employed in clinical and forensic settings. However, we still lack a cost-effective, sensitive, medium-scale method for the analysis of hundreds to thousands of user-defined CpGs suitable for minute DNA input amounts (...
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MDPI AG
2025-03-01
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| Series: | Epigenomes |
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| Online Access: | https://www.mdpi.com/2075-4655/9/1/8 |
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| author | Roy B. Simons Hieab H. H. Adams Manfred Kayser Athina Vidaki |
| author_facet | Roy B. Simons Hieab H. H. Adams Manfred Kayser Athina Vidaki |
| author_sort | Roy B. Simons |
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| description | Background: Epigenetic biomarkers, particularly CpG methylation, are increasingly employed in clinical and forensic settings. However, we still lack a cost-effective, sensitive, medium-scale method for the analysis of hundreds to thousands of user-defined CpGs suitable for minute DNA input amounts (<10 ng). In this study, motivated by promising results in the genetics field, we investigated single-molecule molecular inversion probes (smMIPs) for simultaneous analysis of hundreds of CpGs by using an example set of 514 age-associated CpGs (Zhang model). Methods: First, we developed a novel smMIP design tool to suit bisulfite-converted DNA (Locksmith). Then, to optimize the capture process, we performed single-probe capture for ten selected, representative smMIPs. Based on this pilot, the full smMIP panel was tested under varying capture conditions, including hybridization and elongation temperature, smMIP and template DNA amounts, dNTP concentration and elongation time. Results: Overall, we found that the capture efficiency was highly probe-(and hence, sequence-) dependent, with a heterogeneous coverage distribution across CpGs higher than the 1000-fold range. Considering CpGs with at least 20X coverage, we yielded robust methylation detection with levels comparable to those obtained from the gold standard EPIC microarray analysis (Pearsons’s r: 0.96). Conclusions: The observed low specificity and uniformity indicate that smMIPs in their current form are not compatible with the lowered complexity of bisulfite-converted DNA. |
| format | Article |
| id | doaj-art-6dfec6ced4644a449bfc857b7acc5836 |
| institution | DOAJ |
| issn | 2075-4655 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Epigenomes |
| spelling | doaj-art-6dfec6ced4644a449bfc857b7acc58362025-08-20T02:42:32ZengMDPI AGEpigenomes2075-46552025-03-0191810.3390/epigenomes9010008Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation AnalysisRoy B. Simons0Hieab H. H. Adams1Manfred Kayser2Athina Vidaki3Department of Genetic Identification, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The NetherlandsDepartment of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The NetherlandsDepartment of Genetic Identification, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The NetherlandsDepartment of Genetic Identification, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The NetherlandsBackground: Epigenetic biomarkers, particularly CpG methylation, are increasingly employed in clinical and forensic settings. However, we still lack a cost-effective, sensitive, medium-scale method for the analysis of hundreds to thousands of user-defined CpGs suitable for minute DNA input amounts (<10 ng). In this study, motivated by promising results in the genetics field, we investigated single-molecule molecular inversion probes (smMIPs) for simultaneous analysis of hundreds of CpGs by using an example set of 514 age-associated CpGs (Zhang model). Methods: First, we developed a novel smMIP design tool to suit bisulfite-converted DNA (Locksmith). Then, to optimize the capture process, we performed single-probe capture for ten selected, representative smMIPs. Based on this pilot, the full smMIP panel was tested under varying capture conditions, including hybridization and elongation temperature, smMIP and template DNA amounts, dNTP concentration and elongation time. Results: Overall, we found that the capture efficiency was highly probe-(and hence, sequence-) dependent, with a heterogeneous coverage distribution across CpGs higher than the 1000-fold range. Considering CpGs with at least 20X coverage, we yielded robust methylation detection with levels comparable to those obtained from the gold standard EPIC microarray analysis (Pearsons’s r: 0.96). Conclusions: The observed low specificity and uniformity indicate that smMIPs in their current form are not compatible with the lowered complexity of bisulfite-converted DNA.https://www.mdpi.com/2075-4655/9/1/8age predictioncaptureDNA methylationepigeneticspadlock probessingle-molecule molecular inversion probes |
| spellingShingle | Roy B. Simons Hieab H. H. Adams Manfred Kayser Athina Vidaki Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis Epigenomes age prediction capture DNA methylation epigenetics padlock probes single-molecule molecular inversion probes |
| title | Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis |
| title_full | Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis |
| title_fullStr | Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis |
| title_full_unstemmed | Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis |
| title_short | Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis |
| title_sort | investigating single molecule molecular inversion probes for medium scale targeted dna methylation analysis |
| topic | age prediction capture DNA methylation epigenetics padlock probes single-molecule molecular inversion probes |
| url | https://www.mdpi.com/2075-4655/9/1/8 |
| work_keys_str_mv | AT roybsimons investigatingsinglemoleculemolecularinversionprobesformediumscaletargeteddnamethylationanalysis AT hieabhhadams investigatingsinglemoleculemolecularinversionprobesformediumscaletargeteddnamethylationanalysis AT manfredkayser investigatingsinglemoleculemolecularinversionprobesformediumscaletargeteddnamethylationanalysis AT athinavidaki investigatingsinglemoleculemolecularinversionprobesformediumscaletargeteddnamethylationanalysis |