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Impact of SNPs on methylation readouts by Illumina Infinium HumanMethylation450 BeadChip Array: implications for comparative population studies.

ABSTRACT: Infinium HumanMethylation 450 BeadChip Arrays by Illumina (Illumina HM450K) are among the most popular CpG microarray platforms widely used in biological and medical research. Several recent studies highlighted the potentially confounding impact of the genomic variation on the results of methylation studies performed using Illumina's Infinium methylation probes. However, the complexity of SNPs impact on the methylation level measurements (? values) has not been comprehensively described.In our comparative study of European and Asian populations performed using Illumina HM450K, we found that the majority of Infinium probes, which differentiated two examined groups, had SNPs in their target sequence. Characteristic tri-modal or bi-modal patterns of ? values distribution among individual samples were observed for CpGs with SNPs in the first and second position, respectively. To better understand how SNPs affect methylation readouts, we investigated their impact in the context of SNP position and type, and of the Illumina probe type (Infinium I or II).Our study clearly demonstrates that SNP variation existing in the genome, if not accounted for, may lead to false interpretation of the methylation signal differences suggested by some of the Illumina Infinium probes. In addition, it provides important practical clues for discriminating between differences due to the methylation status and to the genomic polymorphisms, based on the inspection of methylation readouts in individual samples. This approach is of special importance when Illumina Infinium assay is used for any comparative population studies, whether related to cancer, disease, ethnicity where SNP frequencies differentiate the studied groups.

SUBMITTER: Daca-Roszak P

PROVIDER: S-EPMC4659175 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Impact of SNPs on methylation readouts by Illumina Infinium HumanMethylation450 BeadChip Array: implications for comparative population studies.

Daca-Roszak Patrycja P Pfeifer Aleksandra A Żebracka-Gala Jadwiga J Rusinek Dagmara D Szybińska Aleksandra A Jarząb Barbara B Witt Michał M Ziętkiewicz Ewa E

BMC genomics 20151125

<h4>Background</h4>Infinium HumanMethylation 450 BeadChip Arrays by Illumina (Illumina HM450K) are among the most popular CpG microarray platforms widely used in biological and medical research. Several recent studies highlighted the potentially confounding impact of the genomic variation on the results of methylation studies performed using Illumina's Infinium methylation probes. However, the complexity of SNPs impact on the methylation level measurements (β values) has not been comprehensively ...[more]

PMID: 26607064

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Project description:****************************************************************** 450k ARRAY EXPANDED ANNOTATION PLATFORM: SEARCH GPL16304 ****************************************************************** Background: Measurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potentially functional changes in various pathological conditions. Illumina Infinium is a relatively inexpensive and user-friendly DNAm microarray platform used by many researchers to measure DNAm on a large scale. However it has been suggested that a subset of probes may give rise to misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest HumanMethylation450 BeadChip array (with >485,000 probes covering 99% of RefSeq genes). Results: Annotation that was added or expanded on includes 1) SNPs documented in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites. Probes with documented SNPs within 10bp of the target site and especially those with documented SNPs at the target CpG, were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG was used to demonstrate how sample genotype can confound the measurement of DNAm. 8.6% of probes were identified as non-specific, in other words, these probes map to multiple locations in silico. DNAm measured from these non-specific probes likely represents a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by alternative CpG classes rather than UCSC islands provides a more distinctive classification system of CpG sites. Finally variable enrichment for tissue-specific differentially methylated probes was noted across CpG classes and gene feature groups, depending on the tissues that were compared. Conclusion: Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450k array. Additionally CpG enrichment classes and to a lesser extent gene feature groups were associated with distinct patterns of DNAm. Thus, we recommend that confounded probes be removed from analyses and that genomic trends be considered in analyses of the Illumina HumanMethylation450 BeadChip. DNAm arrays offer a powerful approach for which thoughtful use of probe content can be utilized to better understand the biological processes affected.

Dataset Information

Impact of SNPs on methylation readouts by Illumina Infinium HumanMethylation450 BeadChip Array: implications for comparative population studies.

Publications

Impact of SNPs on methylation readouts by Illumina Infinium HumanMethylation450 BeadChip Array: implications for comparative population studies.

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