Evaluation of the Infinium Methylation 450K technology

Abstract

Aims: Studies of DNA methylomes hold enormous promise for biomedicine but are hampered by the technological challenges of analyzing many samples cost-effectively. Recently, a major extension of the previous Infinium HumanMethylation27 BeadChip ® (Illumina, Inc. CA, USA), called Infinium HumanMethylation450 (Infinium Methylation 450K; Illumina, Inc. CA, USA) was developed. This upgraded technology is a hybrid of two different chemical assays, the Infinium I and Infinium II assays, allowing (for 12 samples in parallel) assessment of the methylation status of more than 480,000 cytosines distributed over the whole genome. In this article, we evaluate Infinium Methylation 450K on cell lines and tissue samples, highlighting some of its advantages but also some of its limitations. In particular, we compare the methylation values of the Infinium I and Infinium II assays. Materials & methods: We used Infinium Methylation 450K to profile: first, the well-characterized HCT116 wild-type and double-knockout cell lines and then, 16 breast tissue samples (including eight normal and eight primary tumor samples). Absolute methylation values (b-values) were extracted with the GenomeStudio TM software and then subjected to detailed analysis. Results: While this technology appeared highly robust as previously shown, we noticed a divergence between the b-values retrieved from the type I and type II Infinium assays. Specifically, the b-values obtained from Infinium II probes were less accurate and reproducible than those obtained from Infinium I probes. This suggests that data from the type I and type II assays should be considered separately in any downstream bioinformatic analysis. To be able to deal with the Infinium I and Infinium II data together, we developed and tested a new correction technique, which we called 'peak-based correction'. The idea was to rescale the Infinium II data on the basis of the Infinium I data. While this technique should be viewed as an approximation method, it significantly improves the quality of Infinium II data. Conclusion: Infinium 450K is a powerful technique in terms of reagent costs, time of labor, sample throughput and coverage. It holds great promise for the better understanding of the epigenetic component in health and disease. Yet, due to the nature of its design comprising two different chemical assays, analysis of the whole set of data is not as easy as initially anticipated. Correction strategies, such as the peak-based approach proposed here, are a step towards adequate output data analysis. keywoRds: bisulfite-based method n dNA methylation n dNA methylome n epigenetics n epigenomics n Infinium I n Infinium II n Infinium Methylation 450k n peak-based correction

Distinctive topology of age-associated epigenetic drift in the human interactome

“…So far we have focused on methylation changes within promoters, as this region is known to be of regulatory significance. However, it is important to study if results differ had we used other gene regions, which can be assessed using data (36) generated with the more comprehensive Illumina 450k arrays (44). Consistent with our previous result, genes with age-associated changes in the TSS200 region [i.e., within 200 bp of the transcription start site (TSS)] exhibited significantly lower connectivity than their complement.…”

936 Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer

“…29,31,32 Initially we performed a technical validation of the discovery cohort data by pyrosequence analysis of converted DNA. 25,33,34 In common with previous reports and across multiple genes, these analyses confirmed and reinforced the array-derived data. 34,35,36 These analyses also showed that for the majority of regions investigated, methylation extended to include contiguous promoter-associated CpG sites.…”

“…40 b-values range from 0 (unmethylated) to 1 (fully methylated). NIMBL was used to perform 'peak-based' correction, to adjust for potential differences in array probe-type sensitivity previously reported 33 ; all comparative analyses of high-grade tumors to normal bladder controls, were performed on peak-based corrected b-values, as described by us previously. 68 Each array passed quality control assessment based upon the performance of internal controls and the distribution of b-values across all array CpGs.…”

Whole genome DNA methylation signature of HER2-positive breast cancer

“…This C/T polymorphism generated by the bisulfite treatment is genotyped and used to evaluate CpG methylation in the Infinium methylation assay that covers 96% of CGIs and 99% of human RefSeq genes. 47 A total of 200 ng of bisulfite converted DNA (Bs-DNA) was then used for methylation assay using Infinium HumanMethylation450 BeadChip kit according Illumina Infinium HD Methylation protocol. Briefly, this included whole genome amplification (WGA) where Bs-DNA is denatured, neutralized and incubated at 37 °C followed by enzymatic fragmentation, precipitation and resuspension in hybridization buffer.…”