Illumina Methylation Assay

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The Illumina Methylation Assay using the Infinium I platform uses 'BeadChip' technology[ clarification needed ] to generate a comprehensive genome-wide profiling of human DNA methylation. Similar to bisulfite sequencing and pyrosequencing, this method quantifies methylation levels at various loci within the genome. This assay is used for methylation probes on the Illumina Infinium HumanMethylation27 BeadChip (henceforth, 27k [methylation] array). Probes on the 27k array target regions of the human genome to measure methylation levels at 27,578 CpG dinucleotides in 14,495 genes. [1] In 2008, Illumina released the Infinium HumanMethylation450 BeadChip array (“450 K array”), which targets over 450,000 methylation sites. [2] In 2016, the Infinium MethylationEPIC BeadChip ("EPIC") was released, which interrogates over 850,000 methylation sites across the human genome. [3]

Contents

Background

DNA methylation plays a significant role in the epigenetic regulation of chromatin structure, which in the last decade has been recognized to be important in the regulation of gene expression, development and genetic imprinting in vertebrates. [1] Changes in the methylation pattern and level have been shown to contribute to cancer and various developmental diseases. [4] For example, hypermethylation at the promoter CpG islands of a tumour suppressor gene, which in turn leads to its silencing, is frequently associated with tumourgenesis. [4] A large scale measurement of DNA methylation patterns from a wide selection of genes may enable us to understand better the relationships between epigenetic changes and the genesis of different diseases and a better understanding of the role that epigenetics plays in tissue specific differentiation.

Table 1. Methylation Statistics MethylationBeadChipStats.png
Table 1. Methylation Statistics

Material

The Illumina 27k methylation chip contains 27,578 individual CpG sites, spread across 14,495 genes. [1] These genes include RefSeq genes from the NCBI CCDS Database, cancer genes that show differential methylation patterns during their course of progression and microRNA promoters. [5] The markers included in the chip are summarized in Table 1. [5]

Method

Figure 1. Workflow of the Infinium I assay. A single BeadChip accommodates 12 samples. Only one strand at the (same) locus (corresponding to e.g. the same strand, maternal and paternal copies in a diploid individual that is heterozygous for methylation at this particular locus) is depicted here. In this example, single-base extension was successful for the two dideoxynucleotides containing adenine, corresponding to one methylated and one unmethylated allele in the original (i.e., before bisulphite conversion) genome. Illuminamethylationworkflow.png
Figure 1. Workflow of the Infinium I assay. A single BeadChip accommodates 12 samples. Only one strand at the (same) locus (corresponding to e.g. the same strand, maternal and paternal copies in a diploid individual that is heterozygous for methylation at this particular locus) is depicted here. In this example, single-base extension was successful for the two dideoxynucleotides containing adenine, corresponding to one methylated and one unmethylated allele in the original (i.e., before bisulphite conversion) genome.

For the Infinium I assay chemistry technology, the process is outlined in Figure 1.
Bisulfite treatment
Approximately 1 μg of genomic DNA is used in bisulfite conversion to convert the unmethylated cytosine into uracil. The product contains unconverted cytosine where they were previously methylated, but cytosine converted to uracil if they were previously unmethylated.

Whole-genomic DNA amplification
The bisulfite treated DNA is subjected to whole-genome multiple displacement amplification via random hexamer priming and Φ29 DNA polymerase, which has a proofreading activity resulting in error rates 100 times lower than the Taq polymerase. The products are then enzymatically fragmented, [1] purified from dNTPs, primers and enzymes, and applied to the chip. [6]

Hybridization and Single-base extension
On the chip, there are two bead types for each CpG (or "CG", as per Figure 1) site per locus. Each locus tested is differentiated by different bead types. [1] Both bead types are attached to single-stranded 50-mer DNA oligonucleotides that differ in sequence only at the free end; this type of probe is known as an allele-specific oligonucleotide. One of the bead types will correspond to the methylated cytosine locus and the other will correspond to the unmethylated cytosine locus, which has been converted into uracil during bisulfite treatment and later amplified as thymine during whole-genome amplification. The bisulfite-converted amplified DNA products are denatured into single strands and hybridized to the chip via allele-specific annealing to either the methylation-specific probe or the non-methylation probe. Hybridization is followed by single-base extension with hapten-labeled dideoxynucleotides. The ddCTP and ddGTP are labeled with biotin while ddATP and ddUTP are labeled with 2,4-dinitrophenol (DNP). [7]

Fluorescence staining and scanning of chip
After incorporation of these hapten-labeled ddNTPs, multi-layered immunohistochemical assays are performed by repeated rounds of staining with a combination of antibodies to differentiate the two types. [7] After staining, the chip is scanned to show the intensities of the unmethylated and methylated bead types. (Figure 2). [1] The raw data are analyzed by the proprietary software, and the fluorescence intensity ratios between the two bead types are calculated. For a given individual at a given locus, a ratio value of 0 equals to non-methylation of the locus (i.e., homozygous unmethylated); a ratio of 1 equals to total methylation (i.e., homozygous methylated); and a value of 0.5 means that one copy is methylated and the other is not (i.e., heterozygosity), in the diploid human genome. [1] [5]

Figure 2. Types of data analysis. Dataoutput.png
Figure 2. Types of data analysis.

Analysis of methylation data
The scanned microarray images of methylation data are further analyzed by the system, which normalizes the raw data to reduce the effects of experimental variation, background and average normalization, and performs standard statistical tests on the results. [8] The data can then be compiled into several types of figures for visualization and analysis. Scatter plots are used to correlate the methylation data; bar plots to visualize relative levels of methylation at each site tested; heat maps to cluster the data to compare the methylation profile at the sites tested. Figure 2 shows the different types of results generated.

Advantages and disadvantages

Advantages


Disadvantages

See also

Related Research Articles

<span class="mw-page-title-main">5-Methylcytosine</span> Chemical compound which is a modified DNA base

5-Methylcytosine is a methylated form of the DNA base cytosine (C) that regulates gene transcription and takes several other biological roles. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered. 5-Methylcytosine is incorporated in the nucleoside 5-methylcytidine.

<span class="mw-page-title-main">CpG site</span> Region of often-methylated DNA with a cytosine followed by a guanine

The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG islands.

<span class="mw-page-title-main">DNA methyltransferase</span> Class of enzymes

In biochemistry, the DNA methyltransferase family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.

<span class="mw-page-title-main">Regulation of gene expression</span> Modifying mechanisms used by cells to increase or decrease the production of specific gene products

Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.

<span class="mw-page-title-main">DNA methylation</span> Biological process

DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. In mammals, DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis.

<span class="mw-page-title-main">Epigenome</span> Biological term

In biology, the epigenome of an organism is the collection of chemical changes to its DNA and histone proteins that affects when, where, and how the DNA is expressed; these changes can be passed down to an organism's offspring via transgenerational epigenetic inheritance. Changes to the epigenome can result in changes to the structure of chromatin and changes to the function of the genome. The human epigenome, including DNA methylation and histone modification, is maintained through cell division. The epigenome is essential for normal development and cellular differentiation, enabling cells with the same genetic code to perform different functions. The human epigenome is dynamic and can be influenced by environmental factors such as diet, stress, and toxins.

<span class="mw-page-title-main">Methylation specific oligonucleotide microarray</span> Technique used to map epigenetic methylations in cancer DNA

Methylation specific oligonucleotide microarray, also known as MSO microarray, was developed as a technique to map epigenetic methylation changes in DNA of cancer cells.

For the purpose of DNA replication, the HpaII tiny fragment Enrichment by Ligation-mediated PCR Assay is one of several techniques used for determining whether DNA has been methylated. The technique can be adapted to examine DNA methylation within and around individual genes, or it can be expanded to examine methylation in an entire genome.

<span class="mw-page-title-main">Bisulfite sequencing</span> Lab procedure detecting 5-methylcytosines in DNA

Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.

<span class="mw-page-title-main">Combined bisulfite restriction analysis</span>

Combined Bisulfite Restriction Analysis is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. The technique is a variation of bisulfite sequencing, and combines bisulfite conversion based polymerase chain reaction with restriction digestion. Originally developed to reliably handle minute amounts of genomic DNA from microdissected paraffin-embedded tissue samples, the technique has since seen widespread usage in cancer research and epigenetics studies.

<span class="mw-page-title-main">Bayesian tool for methylation analysis</span>

Bayesian tool for methylation analysis, also known as BATMAN, is a statistical tool for analysing methylated DNA immunoprecipitation (MeDIP) profiles. It can be applied to large datasets generated using either oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq), providing a quantitative estimation of absolute methylation state in a region of interest.

<span class="mw-page-title-main">Reduced representation bisulfite sequencing</span> Methylation process

Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content. Due to the high cost and depth of sequencing to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 to reduce the amount of nucleotides required to sequence to 1% of the genome. The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches.

<span class="mw-page-title-main">Whole genome bisulfite sequencing</span>

Whole genome bisulfite sequencing is a next-generation sequencing technology used to determine the DNA methylation status of single cytosines by treating the DNA with sodium bisulfite before high-throughput DNA sequencing. The DNA methylation status at various genes can reveal information regarding gene regulation and transcriptional activities. This technique was developed in 2009 along with reduced representation bisulfite sequencing after bisulfite sequencing became the gold standard for DNA methylation analysis.

<span class="mw-page-title-main">Epigenome-wide association study</span>

An epigenome-wide association study (EWAS) is an examination of a genome-wide set of quantifiable epigenetic marks, such as DNA methylation, in different individuals to derive associations between epigenetic variation and a particular identifiable phenotype/trait. When patterns change such as DNA methylation at specific loci, discriminating the phenotypically affected cases from control individuals, this is considered an indication that epigenetic perturbation has taken place that is associated, causally or consequentially, with the phenotype.

<span class="mw-page-title-main">GLAD-PCR assay</span>

Glal hydrolysis and Ligation Adapter Dependent PCR assay is the novel method to determine R(5mC)GY sites produced in the course of de novo DNA methylation with DNMTЗA and DNMTЗB DNA methyltransferases. GLAD-PCR assay do not require bisulfite treatment of the DNA.

CpG island hypermethylation is a phenomenon that is important for the regulation of gene expression in cancer cells, as an epigenetic control aberration responsible for gene inactivation. Hypermethylation of CpG islands has been described in almost every type of tumor.

<span class="mw-page-title-main">NOMe-seq</span> NOMe-seq is a nucleosome occupancy and methylome technique.

Nucleosome Occupancy and Methylome Sequencing (NOMe-seq) is a genomics technique used to simultaneously detect nucleosome positioning and DNA methylation... This method is an extension of bisulfite sequencing, which is the gold standard for determining DNA methylation. NOMe-seq relies on the methyltransferase M.CviPl, which methylates cytosines in GpC dinucleotides unbound by nucleosomes or other proteins, creating a nucleosome footprint. The mammalian genome naturally contains DNA methylation, but only at CpG sites, so GpC methylation can be differentiated from genomic methylation after bisulfite sequencing. This allows simultaneous analysis of the nucleosome footprint and endogenous methylation on the same DNA molecules. In addition to nucleosome foot-printing, NOMe-seq can determine locations bound by transcription factors. Nucleosomes are bound by 147 base pairs of DNA whereas transcription factors or other proteins will only bind a region of approximately 10-80 base pairs. Following treatment with M.CviPl, nucleosome and transcription factor sites can be differentiated based on the size of the unmethylated GpC region.

<span class="mw-page-title-main">Epiphenotyping</span> Epiphenotyping is the use of DNA methylation patterns to predict phenotypes.

Epiphenotyping involves studying the relationship between DNA methylation patterns and phenotypic traits in individuals and populations to be able to predict a phenotype from a DNA methylation profile. In the following sections, the background of epiphenotyping, an overview of a general methodology, its applications, advantages, and limitations are covered.

References

  1. 1 2 3 4 5 6 7 8 Weisenberger, DJ. et al. (2008) Comprehensive DNA Methylation Analysis on the Illumina Infinium Assay Platform.
  2. ↑ Infinium® HumanMethylation450 BeadChip
  3. ↑ "Infinium MethylationEPIC Kit | Methylation profiling array for EWAS". www.illumina.com. Retrieved 2020-01-10.
  4. 1 2 DNA methylation in development and human disease. Gopalakrishnan S, Van Emburgh BO, Robertson KD. Mutation Research. 2008 Dec 1;647(1-2):30-8. Epub 2008 Aug 20. Review.
  5. 1 2 3 4 5 6 Illumina: DNA Methylation Analysis: http://www.illumina.com/downloads/DNAMethylationAnalysis_DataSheet.pdf%5B%5D
  6. ↑ Gunderson, KL. et al. A genome-wide scalable SNP genotyping assay using microarray technology. Nature Genetics 37, 549 - 554 (2005).
  7. 1 2 Steemers, FJ. et al. Whole-genome genotyping with the single-base extension assay. Nature methods, Vol. 3, No. 1, Jan, 31 – 33 (2006).
  8. ↑ BeadStudio Methylation Module User Guide. Version 3. Illumina Systems & Software. 2006
  9. ↑ NCBI, Consensus CDS (CCDS) project
  10. 1 2 Staaf, J. et al. Normalization of Illumina Infinium whole-genome SNP data improves copy number estimates and allelic intensity ratios. BMC Bioinformatics 2008, 9:409.
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