Genome-Wide Dna Methylation Profiling Using Infinium^® Assay

Abstract

Aims: Bisulfite sequence analysis of individual CpG sites within genomic DNA is a powerful approach for methylation analysis in the genome. The major limitation of bisulfite-based methods is parallelization. Both array and next-generation sequencing technology are capable of addressing this bottleneck. In this report, we describe the application of Infinium^® genotyping technology to analyze bisulfite-converted DNA to simultaneously query the methylation state of over 27,000 CpG sites from promoters of consensus coding sequences (CCDS) genes. Materials & methods: We adapted the Infinium genotyping assay to readout an array of over 27,000 pairs of CpG methylation-specific query probes complementary to bisulfite-converted DNA. Two probes were designed to each CpG site: a ‘methylated‘ and an ‘unmethylated‘ query probe. The probe design assumed that all underlying CpG sites were ‘in phase‘ with the queried CpG site due to their close proximity. Bisulfite conversion was performed with a modified version of the Zymo EZ DNA Methylation™ kit. Results: We applied this technology to measuring methylation levels across a panel of 14 different human tissues, four Coriell cell lines and six cancer cell lines. We observed that CpG sites within CpG islands (CGIs) were largely unmethylated across all tissues (∼80% sites unmethylated, β < 0.2), whereas CpG sites in non-CGIs were moderately to highly methylated (only ∼12% sites unmethylated, β < 0.2). Within CGIs, only approximately 3–6% of the loci were highly methylated; in contrast, outside of CGIs approximately 25–40% of loci were highly methylated. Moreover, tissue-specific methylation (variation in methylation across tissues) was much more prevalent in non-CGIs than within CGIs. Conclusion: Our results demonstrate a genome-wide scalable array-based methylation readout platform that is both highly reproducible and quantitative. In the near future, this platform should enable the analysis of hundreds of thousands to millions of CpG sites per sample.

KEYWORDS::

• bisulfite
• CCDS
• CpG
• DNA array
• DNA methylation
• Infinium^®

Acknowledgements

We thank our Illumina colleagues in manufacturing for providing oligos and arrays for conducting the described research. We thank the BeadStudio team for the development of the Integrated Genome Viewer, and particularly Ivan Mikoulitch for development of the methylation module enabling analysis of the data. We also thank Eric Allen for helpful suggestions on data analysis.

Financial & competing interests disclosure

Marina Bibikova, Jennie Le, Bret Barnes, Shadi Saedinia-Melnyk, Richard Shen and Kevin L Gunderson are employees of Illumina and may own stock in the company. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.