Illumina Methylation Assay
Encyclopedia
The Illumina
Methylation Assay using the Infinium II platform uses 'BeadChip' technology to generate a comprehensive genome
wide profiling of human DNA methylation
. Similar to bisulfite sequencing
and pyrosequencing
, this method quantifies methylation
levels at specific loci
within the genome
. Although the assay does not encompass the entire human genome
, it can measure methylation level at 27,578 CpG dinucloeotides
in 14,495 genes.
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. Changes in the methylation pattern and level have been shown to contribute to cancer
and various developmental diseases. 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
. A large scale measurement of DNA methylation patterns from a wide selection of gene
s 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 epigenetic plays in tissue specific differentiation
.
Bisulfite treatment
Approximately 1 ug 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 amplification (WGA) via random hexamer priming and Phi29 DNA polymerase
, which has a proofreading activity resulting in error rates 100 times lower than the Taq polymerase
. The products are then enzymatically fragmented, purified from dNTPs, primers and enzymes, and applied to the chip.
Hybridization and Single-base extension
On the chip, there are two bead types for each CpG site per locus. Each locus tested is differentiated by different bead types, there are over 200,000 bead types available. Each of the bead types are attached to a single stranded 50-mer DNA oligonucleotide
s that differ in sequence only at the free end; this type of probe is known as a 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
labelled dideoxynucleotides. The ddCTP is labelled with biotin
while ddATP, ddUTP and ddGTP are labelled with 2,4-dinitrophenol
(DNP)
Fluorescence staining and scanning of chip
After incorporation of these hapten labelled ddNTPs, multi-layered immunohistochemical
assays are performed by repeated rounds of staining with a combination of antibodies to differentiate the two types. After staining, the chip is scanned to show the intensities of the unmethylated and methylated bead types. (Figure 2). The raw data are analyzed by the proprietary software, and the fluorescence intensity ratios between the two bead types are calculated. A ratio value of 0 equals to non-methylation of the locus; a ratio of 1 equals to total methylation; a value of 0.5 means that one copy is methylated and the other is not, in the diploid human genome.
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. 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 map
s to cluster the data to compare the methylation profile at the sites tested. Figure 2 shows the different types of results generated.
Disadvantages
Illumina (company)
Illumina, Inc. is a company incorporated in April 1998 that develops, manufactures and markets integrated systems for the analysis of genetic variation and biological function. Using its technologies, the company provides a line of products and services that serve the sequencing, genotyping and...
Methylation Assay using the Infinium II platform uses 'BeadChip' technology to generate a comprehensive genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
wide profiling of human DNA methylation
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
. Similar to bisulfite sequencing
Bisulfite sequencing
Bisulfite sequencing is the use of bisulfite treatment of DNA to determine its pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied...
and pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...
, this method quantifies methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
levels at specific loci
Locus (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
within the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. Although the assay does not encompass the entire human genome
Human genome
The human genome is the genome of Homo sapiens, which is stored on 23 chromosome pairs plus the small mitochondrial DNA. 22 of the 23 chromosomes are autosomal chromosome pairs, while the remaining pair is sex-determining...
, it can measure methylation level at 27,578 CpG dinucloeotides
CpG site
CpG sites or CG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. "CpG" is shorthand for "—C—phosphate—G—", that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides...
in 14,495 genes.
Background
DNA methylation plays a significant role in the epigeneticEpigenetics
In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name epi- -genetics...
regulation of chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
structure, which in the last decade has been recognized to be important in the regulation of gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
, development
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
and genetic imprinting in vertebrates. Changes in the methylation pattern and level have been shown to contribute to cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
and various developmental diseases. For example, hypermethylation at the promoter CpG islands of a tumour suppressor gene
Tumor suppressor gene
A tumor suppressor gene, or anti-oncogene, is a gene that protects a cell from one step on the path to cancer. When this gene is mutated to cause a loss or reduction in its function, the cell can progress to cancer, usually in combination with other genetic changes.-Two-hit hypothesis:Unlike...
, which in turn leads to its silencing
Gene silencing
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification...
, is frequently associated with tumourgenesis
Carcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
. A large scale measurement of DNA methylation patterns from a wide selection of gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s 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 epigenetic plays in tissue specific differentiation
Cellular differentiation
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...
.
Material
The chip contains 27,578 individual CpG sites, spread across 14,495 genes. 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. The markers included in the chip are summarized in Table 1.Method
The process is outlined in Figure 1.Bisulfite treatment
Approximately 1 ug of genomic DNA is used in bisulfite conversion to convert the unmethylated cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
into uracil
Uracil
Uracil is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine. In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine.Uracil is a common and...
. 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 amplification (WGA) via random hexamer priming and Phi29 DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
, which has a proofreading activity resulting in error rates 100 times lower than the Taq polymerase
Taq polymerase
thumb|228px|right|Structure of Taq DNA polymerase bound to a DNA octamerTaq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated by Thomas D. Brock in 1965...
. The products are then enzymatically fragmented, purified from dNTPs, primers and enzymes, and applied to the chip.
Hybridization and Single-base extension
On the chip, there are two bead types for each CpG site per locus. Each locus tested is differentiated by different bead types, there are over 200,000 bead types available. Each of the bead types are attached to a single stranded 50-mer DNA oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s that differ in sequence only at the free end; this type of probe is known as a Allele specific oligonucleotide
Allele specific oligonucleotide
An allele-specific oligonucleotide is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay...
. 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
Annealing (metallurgy)
Annealing, in metallurgy and materials science, is a heat treatment wherein a material is altered, causing changes in its properties such as strength and hardness. It is a process that produces conditions by heating to above the recrystallization temperature, maintaining a suitable temperature, and...
to either the methylation specific probe or the non-methylation probe . Hybridization is followed by single base extension with hapten
Hapten
A hapten is a small molecule that can elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself...
labelled dideoxynucleotides. The ddCTP is labelled with biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
while ddATP, ddUTP and ddGTP are labelled with 2,4-dinitrophenol
2,4-Dinitrophenol
2,4-Dinitrophenol , C6H4N2O5, is a cellular metabolic poison. It uncouples oxidative phosphorylation by carrying protons across the mitochondrial membrane, leading to a rapid consumption of energy without generation of ATP....
(DNP)
Fluorescence staining and scanning of chip
After incorporation of these hapten labelled ddNTPs, multi-layered immunohistochemical
Immunostaining
Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941...
assays are performed by repeated rounds of staining with a combination of antibodies to differentiate the two types. After staining, the chip is scanned to show the intensities of the unmethylated and methylated bead types. (Figure 2). The raw data are analyzed by the proprietary software, and the fluorescence intensity ratios between the two bead types are calculated. A ratio value of 0 equals to non-methylation of the locus; a ratio of 1 equals to total methylation; a value of 0.5 means that one copy is methylated and the other is not, in the diploid human genome.
Analysis of methylation data
The scanned microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
images of methylation data are further analyzed by the system, which normalizes
Normalization (statistics)
In one usage in statistics, normalization is the process of isolating statistical error in repeated measured data. A normalization is sometimes based on a property...
the raw data to reduce the effects of experimental variation, background and average normalization, and performs standard statistical tests on the results. The data can then be compiled into several types of figures for visualization and analysis. Scatter plots
Scatterplot
A scatter plot or scattergraph is a type of mathematical diagram using Cartesian coordinates to display values for two variables for a set of data....
are used to correlate the methylation data; bar plots to visualize relative levels of methylation at each site tested; heat map
Heat map
A heat map is a graphical representation of data where the values taken by a variable in a two-dimensional table are represented as colors. Fractal maps and tree maps both often use a similar system of color-coding to represent the values taken by a variable in a hierarchy...
s 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- No PCR is required, which means that there will be no selective bias towards shorter fragments.
- Ability to survey up to 12 samples per chip allows for high throughput processing.
- Allows integration of data between other platforms such as gene expressionGene expressionGene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
and microRNA profiling. - The method looks at ~2 CpG sites per CpG island, providing genome-wide coverage of methylation patterns
Disadvantages
- Not every gene annotated in the NCBINational Center for Biotechnology InformationThe National Center for Biotechnology Information is part of the United States National Library of Medicine , a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper...
database was included in the design of this assay, which covers 14,495 genes out of the 17,052 GeneIDs present to date (Human build 36.3). - According to Staaf et al. (2008), “the Infinium II assay seemed to have dye intensity biases between the two channels used in fluorescence detection. Furthermore, this bias was not eliminated even after the data had gone through normalization algorithms used in the BeadStudio software.” This concern, while valid for the GoldenGate methylation assay, is not relevant to the HumanMethylation27k chips, where both probes in a pair extend and fluoresce within the same channel.