Cited by 113Epigenetics and DNA MethylationGene expression and cancer classificationGenetic Syndromes and Imprinting

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Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc.

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Roessler, Jessica, Ammerpohl, Ole, Gutwein, Jana, Hasemeier, Britta, Anwar, Sumadi Lukman, Kreipe, Hans, Lehmann, Ulrich·2012·BMC Research Notes

DOI 10.1186/1756-0500-5-210PMC PMC3420245

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Abstract

Background: The newly released 450k DNA methylation array from Illumina, Inc. offers the possibility to analyze more than 480,000 individual CpG sites in a user friendly standardized format. In this study the relationship between the β-values provided by the Illumina, Inc. array for each individual CpG dinucleotide and the quantitative methylation levels obtained by pyrosequencing were analyzed. In addition, the representation of microRNA genes and imprinted loci on the Illumina, Inc. array was assessed in detail. Genomic DNA from 4 human breast cancer cell lines (IPH-926, HCC1937, MDA-MB-134, PMC42) and 18 human breast cancer specimens as well as 4 normal mammary epithelial fractions was analyzed on 450k DNA methylation arrays. The β-values for 692 individual CpG sites from 62 different genes were cross-validated using conventional quantitative pyrosequencing. Findings: The newly released 450k methylation array from Illumina, Inc. shows a high concordance with quantitative pyrosequencing if identical CpG sites are analyzed in cell lines (Spearman r = 0.88, p ≪ 0.0001), which is somewhat reduced in primary tumor specimens (Spearman r = 0.86, p ≪ 0.0001). 80.7% of the CpG sites show an absolute difference in methylation level of less than 15 percentage points. If different CpG sites in the same CpG islands are targeted the concordance is lower (r = 0.83 in cell lines and r = 0.7 in primary tumors). The number of CpG sites representing microRNA genes and imprinted loci is very heterogeneous (range: 1 – 70 CpG sites for microRNAs and 1 – 288 for imprinted loci). Conclusions: The newly released 450k methylation array from Illumina, Inc. provides a genome-wide quantitative representation of DNA methylation aberrations in a convenient format. Overall, the congruence with pyrosequencing data is very good. However, for individual loci one should be careful to translate the β-values directly into percent methylation levels.

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Figure 1 Cross-validation of 450k methylation array and quantitative pyrosequencing. Shown are scatter plots for the methylation values obtained by pyrosequencing (x-axis) and the β-values obtained from the 450k array for identical CpG sites. A) cell lines (Spearman r = 0.88, linear regression r2 = 0.89, slope: 0.89, 95% confidence interval: 0.86 – 0.93), B) primary human tumor tissues (Spearman r = 0.85, linear regression r2 = 0.77, slope: 0.86, 95% confidence interval: 0.81 – 0.91). Each dot in the figure represents the comparison of % methylation according to pyrosequencing versus the β-value for a single CpG site in one sample. C) and D) corresponding Bland-Altman plots. The difference between both methods for every individual measurement is plotted against the mean of both methods. The mean of the differences +/− two times the standard deviation denotes the 95% range for the limits of agreement (marked by the horizontal dotted lines). For the construction of these plots the β-values have been multiplied by the factor 100 in order to obtained data sets of the same size range. The coordinates of the individual CpG sites analyzed in this comparison are compiled in Additional file 3CC BY · PubMed Central

Figure 2 Representation of theSNRNPlocus on the 450k methylation array. This figure shows the organization of the SNRPN locus with the four differentially methylated regions (red lollipops). Below each region the number of CpG sites represented on the 450k array is indicated (based on Robertson 2005 [16] and references therein)CC BY · PubMed Central

Figure 3 Comparison of methylation levels for four imprinted loci. The methylation level of GNAS, KvDMR, RB1, and DIRAS3 was measured in four healthy normal mammary tissue samples using pyrosequencing and the 450k methylation array. Shown are the mean values, the error bars indicating the standard deviation. For KvDMR six CpG sites represented on the 450k array are measured by the pyrosequencing assay. For the remaining three loci only one CpG site was also measured by the pyrosequencing assay. In addition the data from Supplementary Table S2 from Woodfine et al. [17] are incorporated. These data are also obtained by pyrosequencing from 3 or 6 normal mammary tissue specimens. No standard deviations are reported for these dataCC BY · PubMed Central

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Authors: Roessler, J., Ammerpohl, O., Gutwein, J., Hasemeier, B., Anwar, S. L., Kreipe, H., & Lehmann, U.
Affiliations: Medizinische Hochschule Hannover (DE)
University Hospital Schleswig-Holstein (DE)
Corresponding author: Lehmann, Ulrich
ORCID: Ammerpohl, Ole
Anwar, Sumadi Lukman
Kreipe, Hans
Lehmann, Ulrich
Venue: BMC Research Notes
Publisher: Springer Science and Business Media LLC
Published: 2012-04-30
Volume: 5
Issue: 1
Pages: 210-210
ISSN: 1756-0500 (ISSN-L)
DOI: 10.1186/1756-0500-5-210
PMC: PMC3420245
OpenAlex: W2097998761
Cited by: 112
References: 24
Citations by year: 2026: 3 · 2024: 1 · 2023: 1 · 2022: 4 · 2021: 11 · 2020: 2 · 2019: 9 · 2018: 10 · 2017: 10 · 2016: 15 · 2015: 18 · 2014: 9 · 2013: 15 · 2012: 4
FWCI: 5.92
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by
Locations: BMC Research Notes (publishedVersion, cc-by)
PubMed Central (publishedVersion, cc-by)
PubMed (publishedVersion)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders: Deutsche Forschungsgemeinschaft (SFB TRR77, Project B1, TRR77)
Deutsche Krebshilfe
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Syndromes and Imprinting
Keywords: DNA methylation, 450k methylation array, pyrosequencing, CpG sites, breast cancer, microRNA genes, genomic imprinting, imprinted loci, CpG islands, mammary epithelial cells
MeSH terms: Breast Neoplasms, Female, Humans, Reproducibility of Results, Sequence Analysis, DNA, Genomic Imprinting, DNA Methylation, Base Pairing, Cell Line, Tumor, snRNP Core Proteins, Genetic Loci

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Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc.

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