Abstract
BackgroundThe Illumina Infinium HumanMethylation450 BeadChip and its successor, Infinium MethylationEPIC BeadChip, have been extensively utilized in epigenome-wide association studies. Both arrays use two fluorescent dyes (Cy3-green/Cy5-red) to measure methylation level at CpG sites. However, performance difference between dyes can result in biased estimates of methylation levels.ResultsHere we describe a novel method, called REgression on Logarithm of Internal Control probes (RELIC) to correct for dye bias on whole array by utilizing the intensity values of paired internal control probes that monitor the two color channels. We evaluate the method in several datasets against other widely used dye-bias correction methods. Results on data quality improvement showed that RELIC correction statistically significantly outperforms alternative dye-bias correction methods. We incorporated the method into the R package ENmix, which is freely available from the Bioconductor website (https://www.bioconductor.org/packages/release/bioc/html/ENmix.html).ConclusionsRELIC is an efficient and robust method to correct for dye-bias in Illumina Methylation BeadChip data. It outperforms other alternative methods and conveniently implemented in R package ENmix to facilitate DNA methylation studies.
Highlights
The Illumina Infinium HumanMethylation450 BeadChip and its successor, Infinium MethylationEPIC BeadChip, have been extensively utilized in epigenome-wide association studies
A typical plot is shown in Additional file 1: Figure S1, which is for a normal breast tissue sample from [4] (GEO accession number: GSM815146). This motivates the new method, REgression on Logarithm of Internal Control probes (RELIC), which first performs a regression on the logarithms of the intensity values of the normalization control probes to derive a quantitative relationship between red and green channels, and uses the relationship to correct for dye-bias on intensity values for whole array
Dye-bias correction is more important for Infinium II probes, it is difficult to compare intensity distributions between red and green channels for Infinium II probes because each channel is for a specific methylation state and the overall quantitative distribution of methylated and unmethylated alleles would not be expected to be the same in every sample
Summary
The Illumina Infinium HumanMethylation450 BeadChip and its successor, Infinium MethylationEPIC BeadChip, have been extensively utilized in epigenome-wide association studies Both arrays use two fluorescent dyes (Cy3-green/Cy5-red) to measure methylation level at CpG sites. The Illumina Infinium HumanMethylation450 BeadChip provides methylation measurements at more than 485,000 individual CpG sites [2], and its successor MethylationEPIC BeadChip provides almost twice as many sites (>850,000) Both arrays are two-color channel (Cy3green/Cy5-red) microarrays and employ two chemical assays (Infinium I and Infinium II). The Illumina method first divides all intensity values in each color channel by the average intensity value of the internal normalization control probes for that channel, and rescales the intensity values using the first sample as the referent. Another method implemented in the Bioconductor package methylumi [12] is
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