Abstract
BackgroundAs the most stable and experimentally accessible epigenetic mark, DNA methylation is of great interest to the research community. The landscape of DNA methylation across tissues, through development and in disease pathogenesis is not yet well characterized. Thus there is a need for rapid and cost effective methods for assessing genome-wide levels of DNA methylation. The Illumina Infinium HumanMethylation450 (450K) BeadChip is a very useful addition to the available methods for DNA methylation analysis but its complex design, incorporating two different assay methods, requires careful consideration. Accordingly, several normalization schemes have been published. We have taken advantage of known DNA methylation patterns associated with genomic imprinting and X-chromosome inactivation (XCI), in addition to the performance of SNP genotyping assays present on the array, to derive three independent metrics which we use to test alternative schemes of correction and normalization. These metrics also have potential utility as quality scores for datasets.ResultsThe standard index of DNA methylation at any specific CpG site is β = M/(M + U + 100) where M and U are methylated and unmethylated signal intensities, respectively. Betas (βs) calculated from raw signal intensities (the default GenomeStudio behavior) perform well, but using 11 methylomic datasets we demonstrate that quantile normalization methods produce marked improvement, even in highly consistent data, by all three metrics. The commonly used procedure of normalizing betas is inferior to the separate normalization of M and U, and it is also advantageous to normalize Type I and Type II assays separately. More elaborate manipulation of quantiles proves to be counterproductive.ConclusionsCareful selection of preprocessing steps can minimize variance and thus improve statistical power, especially for the detection of the small absolute DNA methylation changes likely associated with complex disease phenotypes. For the convenience of the research community we have created a user-friendly R software package called wateRmelon, downloadable from bioConductor, compatible with the existing methylumi, minfi and IMA packages, that allows others to utilize the same normalization methods and data quality tests on 450K data.
Highlights
As the most stable and experimentally accessible epigenetic mark, DNA methylation is of great interest to the epigenetics research community
Allele-specific single-base extension of the probes incorporates a fluorescent label for detection. For both BeadChips the customary index of DNA methylation fraction at a specific CpG site is calculated as β = M/(M + U + α) where M and U are methylated and unmethylated signal intensities and α is an arbitrary offset intended to stabilize β values where fluorescent intensities are low
Conscious of the need to minimize technical variation within datasets while retaining as much information as possible, we explored three sets of probes which we expected to provide performance metrics that could be used to evaluate processing and normalization methods: probes in imprinted differentially methylated region (iDMR), single nucleotide polymorphism (SNP) probes, and CpG sites on the X-chromosome
Summary
As the most stable and experimentally accessible epigenetic mark, DNA methylation is of great interest to the research community. We have taken advantage of known DNA methylation patterns associated with genomic imprinting and X-chromosome inactivation (XCI), in addition to the performance of SNP genotyping assays present on the array, to derive three independent metrics which we use to test alternative schemes of correction and normalization. These metrics have potential utility as quality scores for datasets. The 5th letter (column 3) specifies whether the dye-bias correction was performed (i.e. quantile normalization of M against U), with ‘s’ indicating dye bias correction applied to Type I and Type II probes separately, ‘t’ indicating dye bias correction applied to Type I and Type II probes together and ‘n’ indicating no dye bias correction
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