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

Abstract

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.

Keywords: CpGs; DNA methylation; Illumina microarray; SNPs; cross-reactive probe; oligonucleotide probe; polymorphic CpG.

Figure 1. Enrichment of high identity matches on sex chromosomes for autosomal-targeting probes with significant sex methylation differences. (A) Distribution of Infinium I and (B) Infinium II probes mapped to the sex chromosomes. BLAT was performed on autosomal probes against the in silico bisulfite-converted sex chromosomes. Only the best BLAT match for each probe was used to determine the number of bases that matched (x-axis). The significance of sex-associated methylation difference is based on methylation profiles derived from ARCTIC control cohorts.

Figure 2. Methylation profile graph of SEPHS1P locus where several probes map onto chromosome Y and one probe targets a polymorphic CpG. The colored bars represent the methylation profile across all controls, females on the left of the dashed line, male on the right. The bottom dot plot shows the significance of the male/female methylation differences (open circles are p-values < 10^-12). All probes with p-values < 10^-12 map onto chromosome Y. cg05455372 targets a polymorphic CpG cytosine (rs2863984), which has an allele frequency of 0.42 according to 1000 Genomes data. The schematic representations of probes targeting cg04462931 and cg05455372 are shown in Figure S2.

Figure 3. Polymorphic CpG methylation can reflect underlying SNP genotypes. (A) C/T SNP located at the cytosine position of a polymorphic CpG targeted by an Infinium II probe. The C allele was detected as a methylated allele, while the T allele was detected as an unmethylated allele. (B) A/G SNP located at the guanine position of a polymorphic CpG targeted by an Infinium I probe. The A allele transforms the locus into a non-CpG, which on one hand can lead to true loss of methylation and on the other hand can prohibit single base extension from occurring due to loss of hybridization at the SNP location. The low detection count of methylation calls for the AA genotype was the result of loss of hybridization leading to low signal intensity.