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DNA Methylation Profiling
Marina Bibikova, Ph.D., and Jian-Bing Fan, Ph.D.
Introduction
The study of epigenetic changes on a genome-wide scale, termed epigenomics, is a rapidly growing field. With the advent of microarray technology, the elucidation of such changes can be monitored genome-wide, enabling scientists to study epigenomics of systems at the organism level.
Figure 1. For A-E: a) Only the top strand of the gDNA sequence of interest is shown. If other CpG sites are present in close vicinity of the target CpG site, it is assumed that they have the same methylation status as the site of interest. b) Through a bisulfite conversion step, unmethylated cytosines are converted to uracils, while the methylated cytosines remain unchanged. c) For each CpG site, two pairs of probes are designed: an allele-specific oligo (ASO in gold) and locus-specific oligo (LSO in green) probe pair for the methylated state of the CpG site and a corresponding ASO-LSO pair for the unmethylated state. Pooled oligos anneal to the target sequence. All loci are assayed simultaneously. d) Extension occurs from the matched ASO toward the LSO. e) Ligation (purple) of the extended ASOs to their corresponding LSOs creates PCR templates. The ligated products are then amplified by PCR using fluorescently labeled common primers, and hybridized to a bead array bearing the complementary address sequences. For F: The Illumina code (blue) contained within the LSO is hybridized to a complementary sequence on the bead array (f). Two fluors are then used to distinguish between methylated and unmethylated loci. Locus one above shows an unmethylated site, locus two hemimethylated and locus three fully methylated. Click to enlarge. |
Epigenetic modifications, specifically those altering DNA methylation, have been well documented. DNA methylation affects gene expression patterns and is a component of altered epigenetic profiles.(1) Epigenetic modifications do not affect the genetic code, but affect gene transcriptional regulation and can be heritable.(2-5) These modifications (either hypo- or hypermethylation) that affect gene expression have been used as markers for cancer progression or tumor identification.(6-9) Global hypomethylation and promoter-specific hypermethylation are considered to be associated with conditions such as aging, cancer, and atherosclerosis.(10-15) It has also been shown that dietary deficiencies such as those for B12 can affect methylation status.
An assay for methylation
The GoldenGate Genotyping Assay (Illumina, Inc., San Diego, CA) has been adapted for DNA methylation detection, based on “genotyping” bisulfite-converted genomic DNA. (Figure 1) Unmethylated cytosines are converted to uracils when DNA is treated with bisulfite, while methylated cytosines remain unchanged.
Illumina’s BeadArray technology combines a miniaturized, bead-based array platform with a high level of assay multiplexing. The assay procedure is similar to that described previously for standard SNP genotyping(16) with a few modifications.(17) A four-probe design scheme is used to differentiate between methylated and unmethylated alleles of a target CpG site. Two pairs of allele-specific (ASO) and locus-specific (LSO) oligonucleotides correspond to the methylated or un-methylated sequences respectively. The assay procedure includes allele-specific extension and ligation (Figure 1c-1e), and PCR amplification using universal primers. The resulting products are then hybridized to a bead array at sites bearing complementary address sequences (Figure 1f). These hybridized targets contain a fluorescent label that denotes a methylated or unmethylated state for a given locus.
Methylation status of the interrogated CpG site is then calculated as the ratio of fluorescent signal from one allele relative to the sum of both methylated and unmethylated alleles. This value, also known as b value, ranges from 0 (unmethylated) to 1 (fully methylated).
The GoldenGate Assay uses several different control types to ensure data quality, and each bead type is represented with an average 30-fold redundancy to further enhance accuracy.
Reproducibility
Using the BeadArray platform and GoldenGate Assay for Methylation, reproducible DNA methylation profiles can be obtained between technical replicates with an average R2 of 0.98 0.02. The standard deviation of the b-value obtained for all the 1536 CpG sites across four replicates was less than 0.06 in 99% of cases.
Validation by methylation-specific PCR and bisulfite sequencing
Figure 2. Correct separation is observed for methylation profiles of seven DNA samples from normal tissues and 17 colon, breast, lung, and prostate cancer cell lines based on 64 cancer-specific methylation markers. Green, yellow, and red colors correspond to low, medium, and high methylation levels, respectively. Source: Bibikova, M. et. al. High-throughput DNA methylation profiling using universal bead arrays.” Genome Res 16:383-393 (2006).
Copyright 2006, Cold Spring Harbor Laboratory Research.Click to enlarge |
Methylation-specific PCR (MSP) has been widely used to monitor methylation status of individual genes.(18,19) MSP was used to confirm the methylation measurements generated by the BeadArray. MSP primers that are specific to either methylated or unmethylated DNA were designed to target corresponding CpG sites within the promoter regions of CFTR, DBC1, DLK1, EYA4 and NPY genes. Bisulfite-treated genomic DNAs derived from normal lung tissue and cancer cell lines were analyzed using real-time MSP. Of the 35 MSP data points, 34 were highly concordant with the methylation status determined by the GoldenGate Assay analysis with a Spearman correlation coefficient, r = 0.89.(17) Bisulfite sequencing was also used to validate the BeadArray methylation data. A strong correlation was observed between sequencing results and BeadArray-based methylation analysis (Spearman coefficient r= 0.7). These results suggest that the GoldenGate Assay for Methylation can reliably detect methylation differences in clinical samples and that the assay can be used for both marker discovery and validation.
Methylation profiles in cancer cell lines
As an additional feasibility test, we profiled a panel of 17 colon, breast, lung and prostate cancer cell lines as well as 7 normal tissues and cell lines. Sixteen CpG sites for distinguishing cancer from normal and 48 CpG sites for distinguishing individual cancer types were identified. All cancer samples separated as expected from normal samples by hierarchical clustering with Ward's linkage method and correlation-based distance metric. (Figure 2) The data correlated well with previous cell line methylation profiling results. For example, complete methylation of CpG sites within the GSTP1 gene was observed in prostate cancer cell line LNCaP as previously reported.(20) These results support the observation that hypermethylation occurs in the DNA of cancer cells.
Methylation profiling of human embryonic stem cells
In a recent study,(21) we assessed the methylation profile of HES cells, somatic stem cells, differentiated cells, and cancer cell lines at the same 1536 CpG sites described previously in this Application Note, us
Figure 3A shows that all HES cells were easily distinguished from all other cell lines; these pluripotent cell lines formed one tight cluster that was clearly separated from other clusters. A profile of the 25 most significant CpG sites (from 23 genes) selected by t-test separated pluripotent, undifferentiated HES cells from differentiated normal tissue and cell lines.
Summary
Figure 3. a) Three classes of samples formed well-defined subclusters based on their methylation profiles. These samples include cancer cell lines, differentiated and somatic stem cells, and human embryonic stem cell lines. b) Female samples and male samples co-clusted as expected using methylation data from 36 CpG sites from six X-linked housekeeping genes. Source: Bibikova, M. et. al. Human embryonic stem cells have a unique epigenetic signature. Genome Res(/I) 16:1075-1083 (2006). Copyright 2006, Cold Spring Harbor Laboratory Research.
Click to enlarge. |
The Illumina GoldenGate Assay for Methylation, in conjunction with the BeadArray technology allows investigators to cost-effectively survey genome-wide methylation profiles with confidence. The platform combines 96-sample throughput with feature density of up to 1,536 CpG sites. Oligo sets can be customized and optimized with the assistance of Illumina’s Assay Design Tool (ADT) to meet individual experimental goals. This technology should provide insight into epigenetic mechanisms of gene regulation and much needed knowledge to further clarify genetic mechanisms underlying many of today’s most common diseases and normal development.
About the authors
Marina Bibikova, Ph.D. and Jian-Bing Fan, Ph.D. are with Illumina.
More information about methylation profiling is available from:
Illumina, Inc.
800-809-4566
www.illumina.com
References
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21. Bibikova, M. et. al. Human embryonic stem cells have a unique epigenetic signature. Genome Res. 16:1075-1083 (2006).[
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