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Updated: Mar 29, 2026
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Published on: February 24, 2015
Comparing the Efficiency of Bisulfite and Enzymatic DNA Methylation Conversion Methods for Detection of Colorectal
Stellene Vitaro1, Geraldine Laven-Law1, Ganessan Kichenadasse1,2
1Flinders Health and Medical Research Institute, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, Australia.
Background:
Bisulfite conversion remains the gold standard method for DNA methylation analysis but has known limitations. Enzymatic conversion has recently been developed and overcomes some of these pitfalls. This study compared both methods by measuring known colorectal cancer (CRC) methylated biomarkers in colorectal tissues.
Methods:
Colorectal tumors and matched adjacent normal tissues were collected before treatment at surgery from 24 patients with stage I-IV CRC (n = 6 per stage), aged 46-81 years (50% male). Tissue DNA was extracted and converted using bisulfite and enzymatic conversion. Methylation levels of BCAT1, IKZF1, and SEPTIN9 were quantified by methylation specific quantitative PCR (MS-qPCR) as a percentage of the reference gene (ACTB). Biomarker positivity in tissues was tested using a series of percentage methylation thresholds between ≥1% and ≥20%, and percentile-based cutoffs ranging from 50th to 95th percentiles. The Kruskal-Wallis test compared the methylation levels between the conversion methods.
Results:
There were no significant differences in methylation levels between the bisulfite and enzymatic conversion methods for normal tissue DNA (P > 0.05). In tumor tissues, IKZF1 and SEPTIN9 had significantly higher DNA methylation levels with bisulfite conversion compared to enzymatic conversion (P < 0.05), while BCAT1 showed no significant difference (P > 0.05). Across all thresholds, there were no significant differences in colorectal tissue positivity between conversion techniques (P > 0.05), except IKZF1 at the 50th percentile in normal tissues was significantly hypermethylated in enzymatically converted DNA (P < 0.05).
Conclusions:
Bisulfite conversion has higher DNA methylation levels for certain CRC biomarkers in tumor tissues, suggesting over-estimation of methylation that could affect biomarker specificity.
