Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region
Abstract Background Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing....
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | BMC Medical Genomics |
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| Online Access: | https://doi.org/10.1186/s12920-023-01694-6 |
| _version_ | 1797451118170603520 |
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| author | Sara Gombert Kirsten Jahn Hansi Pathak Alexandra Burkert Gunnar Schmidt Lutz Wiehlmann Colin Davenport Björn Brändl Franz-Josef Müller Andreas Leffler Maximilian Deest Helge Frieling |
| author_facet | Sara Gombert Kirsten Jahn Hansi Pathak Alexandra Burkert Gunnar Schmidt Lutz Wiehlmann Colin Davenport Björn Brändl Franz-Josef Müller Andreas Leffler Maximilian Deest Helge Frieling |
| author_sort | Sara Gombert |
| collection | DOAJ |
| description | Abstract Background Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing. Methods We compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment. Results We could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region). Conclusion Based on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d’être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment. |
| first_indexed | 2024-03-09T14:50:17Z |
| format | Article |
| id | doaj.art-53f21eb283434c95be1a2218ade84fa6 |
| institution | Directory Open Access Journal |
| issn | 1755-8794 |
| language | English |
| last_indexed | 2024-03-09T14:50:17Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Medical Genomics |
| spelling | doaj.art-53f21eb283434c95be1a2218ade84fa62023-11-26T14:30:00ZengBMCBMC Medical Genomics1755-87942023-10-0116111210.1186/s12920-023-01694-6Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter regionSara Gombert0Kirsten Jahn1Hansi Pathak2Alexandra Burkert3Gunnar Schmidt4Lutz Wiehlmann5Colin Davenport6Björn Brändl7Franz-Josef Müller8Andreas Leffler9Maximilian Deest10Helge Frieling11Laboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical SchoolLaboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical SchoolLaboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical SchoolLaboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical SchoolDepartment of Human Genetics, Hannover Medical SchoolResearch Core Unit Genomics, Hannover Medical SchoolResearch Core Unit Genomics, Hannover Medical SchoolDepartment of Genome Regulation, Max Planck Institute for Molecular GeneticsDepartment of Genome Regulation, Max Planck Institute for Molecular GeneticsDepartment of Anesthesiology and Intensive Care Medicine, Hannover Medical SchoolLaboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical SchoolLaboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical SchoolAbstract Background Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing. Methods We compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment. Results We could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region). Conclusion Based on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d’être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment.https://doi.org/10.1186/s12920-023-01694-6TRPA1EpigeneticsMethylationBisulfite sequencingNanopore sequencingCas9-mediated PCR-free enrichment, guideRNAs |
| spellingShingle | Sara Gombert Kirsten Jahn Hansi Pathak Alexandra Burkert Gunnar Schmidt Lutz Wiehlmann Colin Davenport Björn Brändl Franz-Josef Müller Andreas Leffler Maximilian Deest Helge Frieling Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region BMC Medical Genomics TRPA1 Epigenetics Methylation Bisulfite sequencing Nanopore sequencing Cas9-mediated PCR-free enrichment, guideRNAs |
| title | Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region |
| title_full | Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region |
| title_fullStr | Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region |
| title_full_unstemmed | Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region |
| title_short | Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region |
| title_sort | comparison of methylation estimates obtained via minion nanopore sequencing and sanger bisulfite sequencing in the trpa1 promoter region |
| topic | TRPA1 Epigenetics Methylation Bisulfite sequencing Nanopore sequencing Cas9-mediated PCR-free enrichment, guideRNAs |
| url | https://doi.org/10.1186/s12920-023-01694-6 |
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