Principles and challenges of genome-wide DNA methylation analysis

Key Points

  • Methylation of cytosine residues at the carbon 5 position occurs naturally in many bacteria, archaea and eukaryotic species, in which it has various roles in protecting the genome from invading genomic parasites or in controlling the expression potential of regions of the genome.

  • DNA methylation is established after DNA synthesis by dedicated enzymes with specific target sequence recognition sites. The uneven distribution of target sites and sample heterogeneity can result in complex DNA methylation patterns.

  • The genomic distribution of DNA methylation encodes important biological information. Hence, techniques for comprehensively describing DNA methylation patterns have been developed.

  • Many standard molecular biology techniques, such as cloning and PCR, erase DNA methylation information, and hybridization does not distinguish between methylated and unmethylated cytosines.

  • There are three different initial treatments of DNA that can be used to reveal DNA methylation: endonuclease digestion, affinity enrichment and bisulphite conversion.

  • The implementation of array hybridization techniques greatly facilitated genome-scale analysis of DNA methylation. Endonuclease-treated or affinity-enriched DNA methods are particularly well suited for array hybridization, whereas bisulphite conversion techniques are not.

  • Next-generation sequencing allows for whole-genome single-base-pair resolution characterization of DNA methylation patterns, particularly as applied to bisulphite-converted DNA.

  • No single technique excels in all aspects. Sample number and characteristics, as well as the desired accuracy, coverage and resolution, influence the choice of technique.

  • DNA methylation is usually measured on a β-distributed absolute scale from 0 to 1, or 0 to 100%, rather than on an infinite scale of log ratios.

  • The unique data distribution characteristics of DNA methylation will require the development of dedicated bioinformatics and computational tools.

  • Single-molecule and nanopore sequencing approaches are likely to usher in the next revolution in high-throughput DNA methylation analysis.

Abstract

Methylation of cytosine bases in DNA provides a layer of epigenetic control in many eukaryotes that has important implications for normal biology and disease. Therefore, profiling DNA methylation across the genome is vital to understanding the influence of epigenetics. There has been a revolution in DNA methylation analysis technology over the past decade: analyses that previously were restricted to specific loci can now be performed on a genome-scale and entire methylomes can be characterized at single-base-pair resolution. However, there is such a diversity of DNA methylation profiling techniques that it can be challenging to select one. This Review discusses the different approaches and their relative merits and introduces considerations for data analysis.

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Figure 1: Sample throughput versus genome coverage.

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Acknowledgements

I am grateful to K. Siegmund and to members of the University of Southern California Epigenome Center for many helpful discussions. P.W.L.'s research is supported by National Cancer Institute grants R01-CA118699 and U24-CA143882 and by the Norris Foundation, the Ovarian Cancer Research Fund, the Canary Foundation, the Entertainment Industry Foundation and the Riley Foundation.

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Peter W. Laird is consultant for Epigenomics, AG, which has a commercial interest in DNA methylation biomarkers.

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Glossary

Transposons

Mobile DNA elements that can relocate within the genome of their hosts.

Restriction–modification system

A set of enzymes found in many bacteria and archaea that protects the host genome from genomic parasites. Restriction–modification systems consist of sequence-specific restriction endonucleases, which target invading DNA, and associated DNA methyltransferases with similar recognition sequences, which protect the host genome from the action of the endonucleases.

Mismatch repair

A DNA-repair pathway that removes mismatched bases and corrects the insertion or deletion of short stretches of (repeated) DNA.

CpG islands

In eukaryotic genomes, regions of several hundred base pairs that are not depleted of CpGs by 5-methylcytosine deamination owing to them being unmethylated in the germ line. They often overlap transcription start sites. Most definitions of CpG islands set a minimum length (for example, 200 or 500 bp), a minimum observed:expected CpG ratio (for example, greater than 0.6 or 0.65) and a minimum GC content (for example, 50% or 55%).

Isoschizomers

Pairs of structurally distinct restriction enzymes with the same recognition sequence and the same cleavage positions.

Neoschizomers

Pairs of structurally distinct restriction enzymes with the same recognition sequence but with different cleavage positions.

Imprinted

A locus with monoallelic expression determined by the parental origin of the allele.

Chromatin immunoprecipitation

A technique that is used to identify the location of DNA-binding proteins and epigenetic marks in the genome. Genomic sequences containing the mark of interest are enriched by binding soluble DNA chromatin extracts (complexes of DNA and protein) to an antibody that recognizes the mark. Related techniques — such as methylated DNA immunoprecipitation — use antibodies to recognize DNA modifications directly.

Array capture

A method for enriching whole genomic DNA for many regions of interest by hybridization to an array containing RNA or DNA sequences complementary to the regions of interest.

Padlock capture

A method for simultaneously capturing and amplifying large numbers of regions of interest from whole genomic DNA. Each padlock probe has two complementary oligonucleotide sequences that flank a region of interest. The sequences are joined by a loop of DNA that ensures efficient joint hybridization and contains sequences for PCR with universal primers.

Solution hybrid selection

A method for enriching whole genomic DNA for many regions of interest by hybridization to a complex library of RNA or DNA sequences in solution, followed by retrieval of the annealed hybrids.

Hemimethylated

Methylation of a residue on one strand within a palindromic target sequence but not of the corresponding residue within the palindromic target sequence on the complementary DNA strand. Not be confused with monoallelic methylation, in which one allele of a locus is methylated in a diploid organism.

β distribution

A continuous probability distribution with an interval between 0 and 1. Two positive parameters, α and β, are used to define β distributions.

Median absolute deviation

A measure of statistical dispersion that is less influenced by outliers and extreme values than standard deviation. It is defined as the median of the collection of absolute deviations from the data set's median.

Quantile normalization

A method for equalizing the total signal intensities and distributions of probe signal strengths among arrays or among colour channels on an array. It sorts all probes by signal strength and then matches probes at each rank position among arrays and forces the values at each rank position to be equal. An identical distribution of probe signal strengths among the arrays or colour channels is obtained.

LOESS normalization

A computationally intensive method in which a polynomial regression is fitted to each point in the data and more weight is given to data nearer the point of interest. It is often applied to hybridization array data to remove differences in global signal intensity among data sets or colour channels.

MA plot

A representation of microarray data in which M (vertical axis) is the intensity ratio between the red (R) and green (G) colour channels (M=log(R/G)) and A (horizontal axis) is the mean intensity (A=(logR+logG)/2). This representation is often used as a basis for normalizing microarray data, with the underlying assumptions that dye bias is dependent on signal intensity, that the majority of probes do not have very different signal intensities among channels and that approximately the same number of probes in each channel have signal intensities that are stronger than the equivalent probes in the other channel.

Targeted indexing

Indexing refers to the incorporation of short sequences as tagged codes during the construction of a sequencing library, followed by the simultaneous parallel sequencing of libraries from many sources. The source of the DNA sequence for each read can be deduced from the index. This technique can be combined with targeted sequencing of regions of interest enriched by hybrid selection.

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Laird, P. Principles and challenges of genome-wide DNA methylation analysis. Nat Rev Genet 11, 191–203 (2010). https://doi.org/10.1038/nrg2732

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