The Application of Next Generation Sequencing in DNA Methylation Analysis
Abstract
:1. Introduction
1.1. DNA methylation
1.2. Methods for DNA methylation analysis
- 1)
- Methylation sensitive restriction enzyme digestion. The genomic DNA can be digested by methylation sensitive restriction enzymes like HpaII and McrBC to discriminate and/or enrich methylated or unmethylated DNA. The methods based on this approach are limited by providing methylation data only at the restriction enzyme recognition sites or adjacent regions.
- 2)
- Affinity purification. The methylated or unmethylated fractions of genomic DNA can be immunoprecipitated by using antibodies against methylated cytosine, methyl-CpG binding domains or other protein domains [32,33,34,35,36]. Using this method, the genome coverage is limited by the composition of the array for hybridization, and the distribution of the potential affinity targets in the genome, e.g. the density of methylated cytosines or CpG sites, which are unevenly distributed in the genome. The exact methylation state of individual CpG sites cannot be determined using this approach.
- 3)
- Bisulfite conversion of DNA. The method is based on the selective deamination of cytosine but not 5-methylcytosine by treatment with sodium bisulfite [37,38]. Briefly, in the presence of sodium bisulfite, all the unmethylated cytosines are chemically converted to uracil, which is amplified as thymine during PCR. In contrast, the methylated cytosines are not converted, such that in the final sequencing result, the 5-methylcytosine will be still detected as cytosine. Therefore, after bisulfite conversion, the methylated and unmethylated cytosines can be distinguished according to the sequence changes (Figure 2). The bisulfite conversion efficiency is critical for the accuracy and the reliability of the results, especially for non-CpG methylation analysis. The incomplete conversion of unmethylated cytosine to uracil or inappropriate conversion of methylcytosine to thymine can cause over- or underestimination of the methylation level [39,40]. It is also noteworthy that the bisulfite conversion technique cannot be used to discriminate the methylated cytosine from 5-hydroxymethylcytosine (5hmC), which has been recently detected in the Purkinje neurons and embryonic stem cells [41,42]. The underlying reason is that after bisulfite conversion, 5hmC is not deaminated to thymine, but converted to cytosine 5-methylenesulfonate, which is read as cytosine during Sanger sequencing [43,44].
2. Application of next generation sequencing methods for DNA methylation analyses
2.1. 454 sequencing based DNA methylation analysis
2.2. Illumina Solexa sequencing based DNA methylation analysis
- 1)
- The reduced representation bisulfite sequencing (RRBS) method was developed and employed to map the methylation status of the murine genome in different cell lines [21]. The principle of the method is to reduce the complexity of genomic DNA by digesting the genomic DNA into small fragments using methylation insensitive restrictive enzymes like MspI, which recognizes CCGG sequence that are enriched in CGIs. After size selection for short fragments, the digested DNA was bisulfite converted and sequenced by Illumina Genome Analyzer. The library generated from MspI digestion was predicted to contain nearly 90% of CGIs in the mouse genome [58]. Recently, this method has been adapted for the DNA methylation profiling in the human genome, especially for the identification of the methylation changes in human clinical samples based on small amounts (30 ng) of genomic DNA [59]. The genomic coverage is not limited to the CGI (50%) and gene core promoters (65%), but other regions e.g. exons, 3’untranslated regions and repetitive elements are included as well [59].
- 2)
- Two similar approaches based on restriction enzymes digestion and Illumina sequencing, but without bisulfite conversion, have been developed for human methylome analysis. One is called Methyl-sensitive cut counting (MSCC) [60]. Here, the methylation sensitive restriction enzyme HpaII, which cuts unmethylated CCGG sequence, is used to digest the genomic DNA and the generated library is sequenced by Illumina sequencing to reveal unmethylated sites [60]. Another similar approach is called methyl-sequencing [61]. Here, the isoschizomers HpaII and MspI are both used to digest the genomic DNA. After adaptor ligation and size selection, Illumina sequencing is used to sequence the library. As HpaII only digest the unmethylated sites and MspI can digest the sites regardless of methylation, the methylation state of single CpG site can be determined by comparing the different reads number from the two libraries [61]. These methods are useful in reducing the complexity of genomic DNA by focusing on the CpG sites in specific sequence context, but on the other hand they cannot provide a methylation map at single base pair resolution.
- 3)
- Based on Illumina sequencing and bisulfite conversion, array capture [62] and Padlock capture [60,63] were developed for the target specific DNA methylation analysis. Hodges et al. developed a method called bisulfite capture based on hybrid selection techniques. An array containing the probes designed to be complementary to the sequence of interest is used to enrich the target sequences from bisulfite converted genomic DNA. Illumina sequencing was used to sequence the fragments eluted from the arrays. The padlock capture strategy was developed by different groups for DNA methylation analysis in target regions [60,63]. Padlock probes were designed to capture the bisulfite converted targeted sequences. Then, they are ligated to form a circularized single strand of DNA in the target region, which can be further amplified and sequenced. Thousands of probes can be designed for the targeted regions like CGIs and the captured fragments can be sequenced in a single run by Illumina Genome Analyzer. For both above mentioned technologies, the capture efficiency of the designed probes, can potentially affect the measurement of DNA methylation state.
- 4)
- Illumina sequencing of bisulfite converted DNA was also used to quantify the DNA methylation level in mouse primordial germ cells at lower coverage that allowed to analyse some global methylation properties like a strong global reduction of DNA methylation in primordial germ cells [26].
2.3. Other NGS sequencing based DNA methylation analysis
3. Discussion
3.1. Statistical issues in bisulfite sequencing DNA methylation analysis
3.2. Methylation analysis in repeats
3.3. Clonal DNA amplification after bisulfite conversion
3.4. Detection of non-CpG methylation
- Analysis of mitochondrial DNA which is not methylated can be included for conversion control. However, this is not bound to chromatin and the DNA sequence is different so it may not be sufficient as control.
- Sequencing depths can be increased. If non-CpG methylation is happening and biologically relevant, it should be observed in several reads at the same cytosine residue. It is essential at the same time to use barcoded adaptors to exclude clonal amplification of the DNA templates giving rise to the independent reads with same non-CpG methylation patterns.
- Results can be reproduced with independent DNA preparations.
- In order to avoid conversion problems related to the primary sequence, recombinant DNA with same sequence can be added to the genomic DNA and analyzed.
- At key positions, methylation may be confirmed by methods not based on bisulfite conversion.
3.5. The challenge of data analysis
Acknowledgements
References and Notes
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Zhang, Y.; Jeltsch, A. The Application of Next Generation Sequencing in DNA Methylation Analysis. Genes 2010, 1, 85-101. https://doi.org/10.3390/genes1010085
Zhang Y, Jeltsch A. The Application of Next Generation Sequencing in DNA Methylation Analysis. Genes. 2010; 1(1):85-101. https://doi.org/10.3390/genes1010085
Chicago/Turabian StyleZhang, Yingying, and Albert Jeltsch. 2010. "The Application of Next Generation Sequencing in DNA Methylation Analysis" Genes 1, no. 1: 85-101. https://doi.org/10.3390/genes1010085