Quantitative comparison of DNA methylation assays for biomarker development and clinical applications

Abstract

DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.

Details matter: the role of genomic location and assay standardization in DNA methylation analyses

“…Lack of standardization of the methylation analysis, in addition to inclusion of different CpG sites for analyses might also be a contributing factor to why the BLUEPRINT consortium recently recommended absolute quantitative analyses over qMSP/MethyLight [25]. Engaging 18 laboratories across 7 countries, they performed a quantitative comparison of the performance of the most widely used methods for DNA methylation analysis.…”

“…Engaging 18 laboratories across 7 countries, they performed a quantitative comparison of the performance of the most widely used methods for DNA methylation analysis. Carefully selected, designed and validated qMSP/MethyLight assays were however acknowledged for the ability to detect small amounts of methylated DNA in an excess of unmethylated DNA in a cost-effective manner [25], in spite of the generally lower correlation between these assays compared with the absolute quantitative assays. Among the locus-specific analyses for DNA methylation assessment, qMSP/ MethyLight is commonly used.…”

Genome-wide epigenomic profiling for biomarker discovery

“…In light of (i) challenges with the experimental setup when using patient material, (ii) costs, and (iii) the extensive computational analysis associated with the exploratory phase of biomarker discovery, genome-wide screens are often performed on relatively small cohorts. Independent of the (statistical) methods used, it is essential to validate (sets of) candidate biomarkers in follow-up studies on large cohorts using targeted epigenetic approaches before potential application in the clinic [85]. …”

“…To narrow down and validate candidate biomarkers, more targeted DNA methylation assays are used on the same or a very similar-sized cohort [101]. Subsequently, the remaining candidate biomarkers are further validated on larger cohorts using targeted DNA methylation assays that are compatible with routine clinical use, for example, by amplicon bisulfite sequencing [85]. Using this powerful workflow, tumors for which prognostic biomarkers have been identified include rectal cancer [102], breast cancer [103], hepatocellular carcinoma [104], and chronic lymphocytic leukemia (CLL) [105, 106].…”

Enabling Personalized Medicine in Europe by the European Commission’s Funding Activities

“…The project partners contributed to the development of new technologies as well as standard operation procedures and data sharing mechanisms that serve as common worldwide standards in epigenomics. Among many important discoveries, the BLUEPRINT researchers elegantly demonstrated the feasibility of epigenetic analysis for clinical diagnostics [33][34][35][36]. The impressive body of knowledge produced by this project is exempli fied, for example, by the major coordinated set of pub lications recently released.…”