A Pilot Head-to-Head Comparison of Two Bisulfite Conversion Kits for NGS-Based DNA Methylation Analysis of Liquid Biopsy Samples from Colorectal Cancer

Given the important role of early detection in improving cancer outcomes, epigenetic alterations, particularly DNA methylation, have emerged as powerful biomarkers for cancer screening and diagnosis. However, methylation profiling of cfDNA (circulating free DNA) presents particular technical challenges because cfDNA is typically present at very low concentrations and is highly fragmented. Bisulfite conversion, although essential for distinguishing methylated from unmethylated cytosines, can further compromise DNA integrity through chemical-induced loss of DNA during purification steps. Inadequate conversion or excessive DNA loss may consequently reduce library complexity, increase DNA duplication rates, and negatively affect mapping efficiency and downstream sequencing quality. Therefore, optimizing bisulfite conversion is particularly important for reliable methylation profiling of low-input cfDNA. The effectiveness of these diagnostic approaches relies on the accurate detection of DNA methylation patterns, which is fundamentally dependent on the efficiency of bisulfite conversion of genomic DNA. This pilot study aims to compare the performance of two widely used bisulfite conversion kits, namely the EpiTect Bisulfite (EpiTect) Kit (Qiagen, Hilden, Germany) and the EZ DNA Methylation (Zymo) Kit (Zymo Research, Irvine, CA, USA), in the context of plasma-derived cfDNA methylation profiling using five samples, including two plasma samples from metastatic colorectal cancer (mCRC) patients collected at the Department of Gastrointestinal Oncology at The University of Texas MD Anderson Cancer Center, in Houston, TX, USA, and two plasma samples from healthy donors (controls) purchased from Innovative Research (Novi, MI, USA), along with one control DNA sample (Corriel NA12878-N88). Our objective was to evaluate and compare their conversion efficiency, mapping efficiency (percentage of mapped and unmapped reads), duplication rate, and distribution of methylated cytosines in CpG, CHG, and CHH contexts. Our results indicate that samples processed using the Zymo kit consistently outperformed those treated with the EpiTect kit across multiple performance metrics. The Zymo-treated samples yielded a higher concentration of cfDNA following bisulfite conversion (125.34 nM vs. 45.60 nM in samples processed with the Epitect kit). Additionally, the Zymo kit achieved a higher average bisulfite conversion efficiency (99.3% vs. 95.7%), as well as a significantly higher average mapping rate (94.86% vs. 72.4%), with fewer unmapped reads and lower duplication rates. Head-to-head comparison of Bismark methylation calls across the same reference regions showed high concordance between the two protocols. The SEPT9 region was strongly hypermethylated with both protocols (100% with EpiTect and approximately 94% with Zymo), whereas the BMP3 region was predominantly unmethylated (approximately 4% with EpiTect and 7% with Zymo). These findings suggest that the Zymo kit demonstrated improved performance under the conditions evaluated for cfDNA methylation analysis compared to the EpiTect protocol. Further investigation using a larger number of samples is needed to validate our findings.