A systematic evaluation of normalization methods and probe replicability using infinium EPIC methylation data
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Evaluation of normalization pipelines and probe reliability on a methylation array; technical assay reproducibility, the polysemy trap rather than the reproducibility of research.
This study examines technical probe replicability and normalization in a biomedical assay, not research reproducibility.
Technical evaluation of methylation-array normalization and probe reliability; assay/lab-method performance, not metaresearch reproducibility.
Abstract
BACKGROUND: The Infinium EPIC array measures the methylation status of > 850,000 CpG sites. The EPIC BeadChip uses a two-array design: Infinium Type I and Type II probes. These probe types exhibit different technical characteristics which may confound analyses. Numerous normalization and pre-processing methods have been developed to reduce probe type bias as well as other issues such as background and dye bias. METHODS: This study evaluates the performance of various normalization methods using 16 replicated samples and three metrics: absolute beta-value difference, overlap of non-replicated CpGs between replicate pairs, and effect on beta-value distributions. Additionally, we carried out Pearson's correlation and intraclass correlation coefficient (ICC) analyses using both raw and SeSAMe 2 normalized data. RESULTS: The method we define as SeSAMe 2, which consists of the application of the regular SeSAMe pipeline with an additional round of QC, pOOBAH masking, was found to be the best performing normalization method, while quantile-based methods were found to be the worst performing methods. Whole-array Pearson's correlations were found to be high. However, in agreement with previous studies, a substantial proportion of the probes on the EPIC array showed poor reproducibility (ICC < 0.50). The majority of poor performing probes have beta values close to either 0 or 1, and relatively low standard deviations. These results suggest that probe reliability is largely the result of limited biological variation rather than technical measurement variation. Importantly, normalizing the data with SeSAMe 2 dramatically improved ICC estimates, with the proportion of probes with ICC values > 0.50 increasing from 45.18% (raw data) to 61.35% (SeSAMe 2).
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The record
- Venue
- Clinical Epigenetics
- Topic
- Epigenetics and DNA Methylation
- Field
- Biochemistry, Genetics and Molecular Biology
- Canadian institutions
- SickKids FoundationHospital for Sick ChildrenUniversity of TorontoHatch (Canada)
- Funders
- Conselho Nacional de Desenvolvimento Científico e TecnológicoFundação de Amparo à Pesquisa do Estado de São Paulo
- Keywords
- Normalization (sociology)Intraclass correlationReplicateStatisticsPearson product-moment correlation coefficientPercentileReproducibilityEPICCorrelationStandard deviationMathematics
- Has abstract in OpenAlex
- yes