qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements
Abstract Background Human DNA methylation profiling offers great promises in various biomedical applications, including ageing, cancer and even forensics. So far, most DNA methylation techniques are based on a chemical process called sodium bisulfite conversion, which specifically converts non-methy...
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BMC
2025-01-01
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| Series: | Epigenetics Communications |
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| Online Access: | https://doi.org/10.1186/s43682-025-00033-3 |
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| author | Faidra Karkala Roy B. Simons Floor Claessens Vivian Kalamara Manfred Kayser Athina Vidaki |
| author_facet | Faidra Karkala Roy B. Simons Floor Claessens Vivian Kalamara Manfred Kayser Athina Vidaki |
| author_sort | Faidra Karkala |
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| description | Abstract Background Human DNA methylation profiling offers great promises in various biomedical applications, including ageing, cancer and even forensics. So far, most DNA methylation techniques are based on a chemical process called sodium bisulfite conversion, which specifically converts non-methylated cytosines into uracils. However, despite the popularity of this approach, it is known to cause DNA fragmentation and loss affecting standardization, while incomplete conversion may result in potential misinterpretation of methylation-based outcomes. Results To offer the community a solution, we developed qBiCo - a novel quality-control method to address the quantity and quality of bisulfite-converted DNA. qBiCo is a 5-plex, TaqMan® probe-based, quantitative (q)PCR assay that amplifies single- and multi-copy DNA fragments of converted and non-converted nature. It estimates four parameters: converted DNA concentration, fragmentation, global conversion efficiency, and potential PCR inhibition. We optimized qBiCo using synthetic DNA standards and assessed it using standard developmental validation criteria, showcasing that qBiCo is reliable, robust and sensitive down to picogram level. We also evaluated its performance by testing decreasing DNA amounts using several commercial bisulfite conversion kits. Depending on the starting DNA quantity, bisulfite-converted DNA recoveries ranged from 8.5 to 100%, conversion efficiencies from 78 to 99.9%, while certain kits highly fragment DNA, demonstrating large variability in their performance. Towards building a prototype tool, we further optimized key functionalities, for example, by replacing the poorest performing single-plex assay and creating a more representative DNA standard. Aiming to scale-up and move towards implementation, we successfully transferred and validated our novel method in six different qPCR platforms from different major manufacturers. Conclusions Overall, with the present study, we offer researchers in the epigenetic field a novel long-awaited QC tool that for the first time allows them to measure key quality and quantity parameters of the most popular DNA conversion process. The tool also enables standardization to prevent inconsistent data and false outcomes in the future, regardless of the downstream experimental analysis of DNA methylation-based research and applications across different fields of biology and biomedicine. |
| format | Article |
| id | doaj-art-13b17ce2fffa48cbb2003439b2a77e7c |
| institution | Kabale University |
| issn | 2730-7034 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Epigenetics Communications |
| spelling | doaj-art-13b17ce2fffa48cbb2003439b2a77e7c2025-01-19T12:11:37ZengBMCEpigenetics Communications2730-70342025-01-015111710.1186/s43682-025-00033-3qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elementsFaidra Karkala0Roy B. Simons1Floor Claessens2Vivian Kalamara3Manfred Kayser4Athina Vidaki5Department of Genetic Identification, Erasmus MC University Medical Center RotterdamDepartment of Genetic Identification, Erasmus MC University Medical Center RotterdamDepartment of Genetic Identification, Erasmus MC University Medical Center RotterdamDepartment of Genetic Identification, Erasmus MC University Medical Center RotterdamDepartment of Genetic Identification, Erasmus MC University Medical Center RotterdamDepartment of Genetic Identification, Erasmus MC University Medical Center RotterdamAbstract Background Human DNA methylation profiling offers great promises in various biomedical applications, including ageing, cancer and even forensics. So far, most DNA methylation techniques are based on a chemical process called sodium bisulfite conversion, which specifically converts non-methylated cytosines into uracils. However, despite the popularity of this approach, it is known to cause DNA fragmentation and loss affecting standardization, while incomplete conversion may result in potential misinterpretation of methylation-based outcomes. Results To offer the community a solution, we developed qBiCo - a novel quality-control method to address the quantity and quality of bisulfite-converted DNA. qBiCo is a 5-plex, TaqMan® probe-based, quantitative (q)PCR assay that amplifies single- and multi-copy DNA fragments of converted and non-converted nature. It estimates four parameters: converted DNA concentration, fragmentation, global conversion efficiency, and potential PCR inhibition. We optimized qBiCo using synthetic DNA standards and assessed it using standard developmental validation criteria, showcasing that qBiCo is reliable, robust and sensitive down to picogram level. We also evaluated its performance by testing decreasing DNA amounts using several commercial bisulfite conversion kits. Depending on the starting DNA quantity, bisulfite-converted DNA recoveries ranged from 8.5 to 100%, conversion efficiencies from 78 to 99.9%, while certain kits highly fragment DNA, demonstrating large variability in their performance. Towards building a prototype tool, we further optimized key functionalities, for example, by replacing the poorest performing single-plex assay and creating a more representative DNA standard. Aiming to scale-up and move towards implementation, we successfully transferred and validated our novel method in six different qPCR platforms from different major manufacturers. Conclusions Overall, with the present study, we offer researchers in the epigenetic field a novel long-awaited QC tool that for the first time allows them to measure key quality and quantity parameters of the most popular DNA conversion process. The tool also enables standardization to prevent inconsistent data and false outcomes in the future, regardless of the downstream experimental analysis of DNA methylation-based research and applications across different fields of biology and biomedicine.https://doi.org/10.1186/s43682-025-00033-3DNA methylationBisulfite conversionEpigeneticsqPCRConversion efficiencyQuality control |
| spellingShingle | Faidra Karkala Roy B. Simons Floor Claessens Vivian Kalamara Manfred Kayser Athina Vidaki qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements Epigenetics Communications DNA methylation Bisulfite conversion Epigenetics qPCR Conversion efficiency Quality control |
| title | qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements |
| title_full | qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements |
| title_fullStr | qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements |
| title_full_unstemmed | qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements |
| title_short | qBiCo: a method to assess global DNA conversion performance in epigenetics via single-copy genes and repetitive elements |
| title_sort | qbico a method to assess global dna conversion performance in epigenetics via single copy genes and repetitive elements |
| topic | DNA methylation Bisulfite conversion Epigenetics qPCR Conversion efficiency Quality control |
| url | https://doi.org/10.1186/s43682-025-00033-3 |
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