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bioRxiv · 10.1101/2025.05.11.653026

A Comparative Assessment of edgeR and methylKit Pipelines for DNA Methylation Detection

Munoa Hoyos, I.·Araolaza, M.·Calzado, I.·Albizuri, M.·Subiran, N.
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Abstract

Despite the improvements in tool development for DNA methylation analysis, there is a lack of a consensus on computational and statistical models used for differentially methylated cytosine (DMC) identification. This variability complicates the interpretation of findings and raises concerns about the reproducibility and biological significance of the detected results. In this regard, the primary objective of this study was to compare the performance, concordance, and biological relevance of edgeR and methylKit tools in detecting DMCs (the first one based on fold change and the second one based on percentage), following morphine exposure model in mouse embryonic stem cells (mESCs). While a different number of total DMCs was identified by each tool, both pipelines detected a global hypomethylation as a result. Genomic analysis revealed a predominant distribution of DMCs in intergenic and intronic regions on one hand, and in open sea regions on the other hand. Despite the differences in sensitivity, both tools demonstrated moderate concordance in DMCs detection ([~]56%) and high concordance in gene level analysis ([~]90%), identifying similar differentially methylated genes (DMGs). Overall, the results underscore the complementary strengths of methylKit and edgeR and highlight the importance of tool selection for epigenetic studies. As a conclusion, integrating both pipelines is recommended for comprehensive analysis, particularly in studies with complex experimental designs.

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Munoa Hoyos, I., Araolaza, M., Calzado, I., Albizuri, M., Subiran, N.. 2025-05-15. A Comparative Assessment of edgeR and methylKit Pipelines for DNA Methylation Detection. https://doi.org/10.1101/2025.05.11.653026

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