Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses
Abstract Background Detection of DNA methylation biomarkers in circulating cell-free DNA (cfDNA) has great clinical potential for cancer management, but there is a high need for method optimization and standardization. Bisulfite conversion of DNA is the gold-standard pre-treatment method for DNA met...
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BMC
2025-06-01
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| Series: | Clinical Epigenetics |
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| Online Access: | https://doi.org/10.1186/s13148-025-01901-4 |
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| author | Stine H. Kresse Evy Marie Thorkildsen Sara Brandt-Winge Heidi Pharo Hege Marie Vedeld Guro E. Lind |
| author_facet | Stine H. Kresse Evy Marie Thorkildsen Sara Brandt-Winge Heidi Pharo Hege Marie Vedeld Guro E. Lind |
| author_sort | Stine H. Kresse |
| collection | DOAJ |
| description | Abstract Background Detection of DNA methylation biomarkers in circulating cell-free DNA (cfDNA) has great clinical potential for cancer management, but there is a high need for method optimization and standardization. Bisulfite conversion of DNA is the gold-standard pre-treatment method for DNA methylation analyses, but causes also DNA fragmentation and loss. Enzymatic conversion of DNA represents a promising alternative due to the more gentle treatment minimizing damage to DNA. The aim of this study was to evaluate and compare enzymatic and bisulfite conversion to identify the best pre-treatment method for detecting DNA methylation biomarkers in cfDNA from plasma using droplet digital PCR (ddPCR). Results The performance of the NEBNext Enzymatic Methyl-seq Kit (both the full kit intended for sequencing and the sub-component NEBNext Enzymatic Methyl-seq Conversion Module) and the EpiTect Plus DNA Bisulfite Kit was evaluated and compared using normal cfDNA and tumor cfDNA samples from colorectal cancer patients. The cytosine conversion efficiency was 99–100% for both enzymatic and bisulfite conversion. Enzymatic conversion resulted in longer DNA fragments with higher peak fragment sizes compared to bisulfite conversion, but the DNA recovery was considerably lower after enzymatic conversion (34–47%) compared to bisulfite conversion (61–81%). For enzymatic conversion, the full kit gave slightly better DNA recovery than the conversion module. A comparison of five magnetic bead brands, as well as several different magnetic bead-to-sample ratios revealed no major improvements in DNA recovery for the enzymatic conversion. DNA methylation of the biomarker BCAT1 was detected at similar rates in parallel tumor cfDNA samples pre-treated with either enzymatic or bisulfite conversion. However, enzymatic conversion resulted in lower number of positive droplets for both target and control ddPCR assays, in line with the lower DNA recovery after conversion. Conclusions Based on a thorough evaluation of enzymatic and bisulfite conversion of cfDNA using ddPCR, bisulfite conversion emerges as the best pre-treatment method due to higher DNA recovery after conversion and higher number of positive droplets in the ddPCR reactions. |
| format | Article |
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| institution | Kabale University |
| issn | 1868-7083 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | BMC |
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| series | Clinical Epigenetics |
| spelling | doaj-art-d199b836930c4cdab22b998aaa51d84b2025-08-20T03:26:43ZengBMCClinical Epigenetics1868-70832025-06-0117111310.1186/s13148-025-01901-4Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analysesStine H. Kresse0Evy Marie Thorkildsen1Sara Brandt-Winge2Heidi Pharo3Hege Marie Vedeld4Guro E. Lind5Department of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalAbstract Background Detection of DNA methylation biomarkers in circulating cell-free DNA (cfDNA) has great clinical potential for cancer management, but there is a high need for method optimization and standardization. Bisulfite conversion of DNA is the gold-standard pre-treatment method for DNA methylation analyses, but causes also DNA fragmentation and loss. Enzymatic conversion of DNA represents a promising alternative due to the more gentle treatment minimizing damage to DNA. The aim of this study was to evaluate and compare enzymatic and bisulfite conversion to identify the best pre-treatment method for detecting DNA methylation biomarkers in cfDNA from plasma using droplet digital PCR (ddPCR). Results The performance of the NEBNext Enzymatic Methyl-seq Kit (both the full kit intended for sequencing and the sub-component NEBNext Enzymatic Methyl-seq Conversion Module) and the EpiTect Plus DNA Bisulfite Kit was evaluated and compared using normal cfDNA and tumor cfDNA samples from colorectal cancer patients. The cytosine conversion efficiency was 99–100% for both enzymatic and bisulfite conversion. Enzymatic conversion resulted in longer DNA fragments with higher peak fragment sizes compared to bisulfite conversion, but the DNA recovery was considerably lower after enzymatic conversion (34–47%) compared to bisulfite conversion (61–81%). For enzymatic conversion, the full kit gave slightly better DNA recovery than the conversion module. A comparison of five magnetic bead brands, as well as several different magnetic bead-to-sample ratios revealed no major improvements in DNA recovery for the enzymatic conversion. DNA methylation of the biomarker BCAT1 was detected at similar rates in parallel tumor cfDNA samples pre-treated with either enzymatic or bisulfite conversion. However, enzymatic conversion resulted in lower number of positive droplets for both target and control ddPCR assays, in line with the lower DNA recovery after conversion. Conclusions Based on a thorough evaluation of enzymatic and bisulfite conversion of cfDNA using ddPCR, bisulfite conversion emerges as the best pre-treatment method due to higher DNA recovery after conversion and higher number of positive droplets in the ddPCR reactions.https://doi.org/10.1186/s13148-025-01901-4Enzymatic conversionEnzymatic Methyl-seqBisulfite conversionDNA methylationLiquid biopsyPlasma |
| spellingShingle | Stine H. Kresse Evy Marie Thorkildsen Sara Brandt-Winge Heidi Pharo Hege Marie Vedeld Guro E. Lind Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses Clinical Epigenetics Enzymatic conversion Enzymatic Methyl-seq Bisulfite conversion DNA methylation Liquid biopsy Plasma |
| title | Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses |
| title_full | Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses |
| title_fullStr | Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses |
| title_full_unstemmed | Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses |
| title_short | Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses |
| title_sort | comparison of enzymatic and bisulfite conversion of circulating cell free tumor dna for dna methylation analyses |
| topic | Enzymatic conversion Enzymatic Methyl-seq Bisulfite conversion DNA methylation Liquid biopsy Plasma |
| url | https://doi.org/10.1186/s13148-025-01901-4 |
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