Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray

Epigenetics, 2013

1,180 citations recorded by the source corpus.

DNA methylation, an important type of epigenetic modification in humans, participates in crucial cellular processes, such as embryonic development, X-inactivation, genomic imprinting and chromosome stability. Several platforms have been developed to study genome-wide DNA methylation. Many investigators in the field have chosen the Illumina Infinium HumanMethylation microarray for its ability to reliably assess DNA methylation following sodium bisulfite conversion. Here, we analyzed methylation profiles of 489 adult males and 357 adult females generated by the Infinium HumanMethylation450 microarray. Among the autosomal CpG sites that displayed significant methylation differences between the two sexes, we observed a significant enrichment of cross-reactive probes co-hybridizing to the sex chromosomes with more than 94% sequence identity. This could lead investigators to mistakenly infer the existence of significant autosomal sex-associated methylation. Using sequence identity cutoffs derived from the sex methylation analysis, we concluded that 6% of the array probes can potentially generate spurious signals because of co-hybridization to alternate genomic sequences highly homologous to the intended targets. Additionally, we discovered probes targeting polymorphic CpGs that overlapped SNPs. The methylation levels detected by these probes are simply the reflection of underlying genetic polymorphisms but could be misinterpreted as true signals. The existence of probes that are cross-reactive or of target polymorphic CpGs in the Illumina HumanMethylation microarrays can confound data obtained from such microarrays. Therefore, investigators should exercise caution when significant biological associations are found using these array platforms. A list of all cross-reactive probes and polymorphic CpGs identified by us are annotated in this paper.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/2aee86d5bf73138010a76536183d896a7a5a3200

Citations in the corpus

• Functional normalization of 450k methylation array data improves replication in large cancer studies (2014, positive): “We therefore remove a set of 30,969 probes that have been shown to cross-hybridize between genomic regions [43].”
• DNA Methylation in Newborns and Maternal Smoking in Pregnancy: Genome-wide Consortium Meta-analysis. (2016, positive): “Also using the MoBa1 cohort, we visually inspected density plots for each of the probes that matched to the list of polymorphic probes from Chen et al. to assess departures from unimodality, including from small numbers of outlier values.”
• Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling (2016, positive): “We followed the written description in [16] to identify regions of potential cross-reactivity using the BLAT alignment tool [45].”
• A Six Months Exercise Intervention Influences the Genome-wide DNA Methylation Pattern in Human Adipose Tissue (2013, neutral): “A comparison of our 1,009 significant CpG sites with Infinium probes reported to cross-react to alternative genomic locations [27] showed only one probe with 50 bases and 14 probes with 49 bases matching to an alternative genomic location.”
• Controlling bias and inflation in epigenome- and transcriptome-wide association studies using the empirical null distribution (2016, neutral): “Ambiguously mapped probes [43], probes with a high detection P value (> 0.”
• SeSAMe: reducing artifactual detection of DNA methylation by Infinium BeadChips in genomic deletions (2018, positive): “The impact of SNPs on the Infinium DNA methylation array measurements has been extensively investigated (13,32), and the exclusion of probes affected by SNPs from analysis is becoming increasingly recognized as an important 2(13) 2(9) 2(5) Total Intensity (Su + Sm) B et a V al u e”
• DNA methylation aging clocks: challenges and recommendations (2019, positive): “7% of the 450k array probes overlap Alu elements, and this small fraction is ~4x more likely to be from the recognized set of technically poor functioning probes [156].”
• Multi-stage differentiation defines melanoma subtypes with differential vulnerability to drug-induced iron-dependent oxidative stress (2018, positive): “Probes excluded from the downstream analysis were probes with poor detection quality, probes mapping to sex chromosomes, probes with known SNPs at the CG site, and 29,233 probes previously shown to be cross-reactive with genes on sex chromosomes (Chen et al., 2013).”
• Comprehensive characterization, annotation and innovative use of Infinium DNA methylation BeadChip probes (2016, positive): “For the third type of influence, one study singled out and excluded all such extension-base SNPs (12).”