biostudies-literatureLin YYBMBFJoachim Herz FoundationGerman Center for Cardiovascular ResearchWilhelm Sander-StiftungHorizon EuropeCardio-Pulmonary InstituteKU LeuvenGerman Cancer AidHorizon 2020German Network for Bioinformatics Infrastructuregkaf970https://www.ebi.ac.uk/biostudies/studies/S-EPMC12539629biostudies-literatureUnknown53(19)DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five whole-genome profiling protocols. As an evaluation reference, we employed accurate locus-specific measurements from our previous beNucleic acids researchPipeline Olympics: continuable benchmarking of computational workflows for DNA methylation sequencing data against an experimental gold standard.PMC125396297011386981Z020010101KD2207A101058427390649896STG/22/024031L0162B2022.010.1031L0101A824110Plass CNordstroem KLipka DBLin YYKensche PRSchonung MGiacomelli GLafrenz PIskar MEsteller MZapatka MWeichenhan DLawerenz CSchulz MHSchlesner MMerkel ABock CGerhauser CChepeleva MWolf SHovestadt VGonzalez-Avalos EHartmann MAssenov YKramer SMucke OToth RWalter JSarnataro ALutsik PTwardziok SBreuer KBuchhalter IThommen FKretzmer HFischer JHoffmann SWilk APetermann FWeiser LfalsePipeline Olympics: continuable benchmarking of computational workflows for DNA methylation sequencing data against an experimental gold standard.DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five whole-genome profiling protocols. As an evaluation reference, we employed accurate locus-specific measurements from our previous be2025-01-01T00:00:00Z2025 Oct2026-05-10T03:23:35.345Z2026-05-10T03:12:29.433ZS-EPMC125396294111857510.1093/nar/gkaf970