Gap hunting to characterize clustered probe signals in Illumina methylation array data

Background: The Illumina 450k array has been widely used in epigenetic association studies. Current quality-control (QC) pipelines typically remove certain sets of probes, such as those containing a SNP or with multiple mapping locations. An additional set of potentially problematic probes are those with DNA methylation distributions characterized by two or more distinct clusters separated by gaps. Data-driven identification of such probes may offer additional insights for downstream analyses. Results: We developed a procedure, termed gap hunting, to identify probes showing clustered distributions. Among 590 peripheral blood samples from the Study to Explore Early Development, we identified 11,007 gap probes. The vast majority (9199) are likely attributed to an underlying SNP(s) or other variant in the probe, although SNP-affected probes exist that do not produce a gap signals. Specific factors predict which SNPs lead to gap signals, including type of nucleotide change, probe type, DNA strand, and overall methylation state. These expected effects are demonstrated in paired genotype and 450k data on the same samples. Gap probes can also serve as a surrogate for the local genetic sequence on a haplotype scale and can be used to adjust for population stratification. Conclusions: The characteristics of gap probes reflect potentially informative biology. QC pipelines may benefit from an efficient data-driven approach that flags gap probes, rather than filtering such probes, followed by careful interpretation of downstream association analyses. Our results should translate directly to the recently released Illumina EPIC array given the similar chemistry and content design.