Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues (original) (raw)
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Comparison of Illumina 450K and EPIC arrays in placental DNA methylation
Epigenetics, 2019
Illumina HumanMethylation450 BeadChip (450K) has been commonly used to investigate DNA methylation in human tissues. Recently, it has been replaced by Illumina HumanMethylationEPIC BeadChip (EPIC) covering over 850,000 CpGs distributed genome-wide. Many consortia have now datasets coming from both arrays and aspire to analyze the two together. The placenta shows a high number of intermediate methylation levels and is often investigated for obstetric/birth outcomes, and potentially for long-term programming in offspring. We performed a systematic comparison between the two arrays using 108 duplicate placental samples from Gen3G birth cohort. We find that placenta shows a high per-sample correlation between the arrays, and higher median correlations at individual CpGs than those reported for blood. We identify 26,340 probes with absolute difference in per cent methylation >10%. We conclude that EPIC and 450K placental data can be combined, and we provide two lists of CpGs that should be excluded to avoid misleading results.
Epigenetics, 2018
Assessing DNA methylation profiles in human blood has become a major focus of epidemiologic inquiry. Understanding variability in CpG-specific DNA methylation over moderate periods of time is a critical first step in identifying CpG sites that are candidates for DNA methylation-based etiologic, diagnostic and prognostic predictors of pathogenesis. Using the Illumina MethylationEPIC [850K] BeadArray, DNA methylation was profiled in paired whole blood samples collected approximately 1 year apart from 35 healthy women enrolled in the Nurses Study II cohort. The median intraclass correlation coefficient (ICC) across all CpG loci was 0.19 [Interquartile Range (IQR) 0.00-0.50]; 74.8% of ICCs were in the low range (0-0.5), 16.9% in the mid-range of ICCs (0.5-0.8), and 8.3% in the high-range of ICCs (0.8-1). ICCs were similar for CpG probes on the 450K Illumina array (median 0.17) and the new probes added to the 850K array (median 0.21). ICCs for CpG loci on the sex chromosomes and known metastable epialleles were high (median 0.71, 0.97, respectively), and ICCs among methylation quantitative trait loci (mQTL) CpGs were significantly higher as compared to non-mQTL CpGs (median 0.73, 0.16, respectively, P < 2 × 10-16). We observed wide variation in DNA methylation stability over a 1-year period. Probes considered non-stable, due to substantial variation over a moderate period of time and with minimal variability across individuals could be removed in large epidemiological studies. Moreover, adjusting for technical variation that arises from using high-dimensional arrays is critical.
Current Advances in DNA Methylation Analysis Methods
BioMed Research International
DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadva...
Genetics research, 2016
DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WB...
Analytical Chemistry, 2005
Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatographyelectrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2′-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2′-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2′-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2′-deoxycytidine to 2′-deoxyguanosine and was determined directly using 2′-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2′-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection. Figure 5. Extracted ion LC-ESI-MS/MS chromatogram of 1 fmol of 5mdC, 2 pmol of dG, and 200 pmol of dG, showing the detection of 1 fmol of 5mdC in DNA hydrolysis mixtures containing either 2 or 200 pmol of dG. The LC-ESI-MS/MS conditions were the same as used in Figure 3. The detection of 5mdC with a transition pair of m/z 242.1/126.3 generated very low background noise (∼20 counts/s), resulting in a LOD of 0.2 fmol.
Disagreement between two common biomarkers of global DNA methylation
Background: The quantification of global DNA methylation has been established in epigenetic screening. As more practicable alternatives to the HPLC-based gold standard, the methylation analysis of CpG islands in repeatable elements (LINE-1) and the luminometric methylation assay (LUMA) of overall 5-methylcytosine content in " CCGG " recognition sites are most widely used. Both methods are applied as virtually equivalent, despite the hints that their results only partly agree. This triggered the present agreement assessments.
Experimental Cell Research, 2006
Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing™. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200-500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer. ava i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s ev i e r. c o m / l o c a t e / yexc r