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Bisulfite-based epityping on pooled genomic DNA provides an accurate estimate of average group DNA methylation.

Docherty SJ, Davis OS, Haworth CM, Plomin R, Mill J - Epigenetics Chromatin (2009)

Bottom Line: Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however, they require relatively large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects.Comparison of data generated from the pooled DNA samples with results averaged across the individual samples comprising each pool revealed highly significant correlations for individual CpG sites across all nine regions, with an average overall correlation across all regions and pools of 0.95 (95% bootstrapped confidence intervals: 0.94 to 0.96).Such an approach can be readily applied to the assessment of disease phenotypes reducing the time, cost and amount of DNA starting material required for large-scale epigenetic analyses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King's College London, De Crespigny Park, Denmark Hill, London, SE5 8AF, UK. spjgsdo@iop.kcl.ac.uk

ABSTRACT

Background: DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however, they require relatively large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. We propose that a high-throughput DNA pooling approach will facilitate the use of emerging methylomic profiling techniques in large samples.

Results: Compared with data generated from 89 individual samples, our analysis of 205 CpG sites spanning nine independent regions of the genome demonstrates that DNA pools can be used to provide an accurate and reliable quantitative estimate of average group DNA methylation. Comparison of data generated from the pooled DNA samples with results averaged across the individual samples comprising each pool revealed highly significant correlations for individual CpG sites across all nine regions, with an average overall correlation across all regions and pools of 0.95 (95% bootstrapped confidence intervals: 0.94 to 0.96).

Conclusion: In this study we demonstrate the validity of using pooled DNA samples to accurately assess group DNA methylation averages. Such an approach can be readily applied to the assessment of disease phenotypes reducing the time, cost and amount of DNA starting material required for large-scale epigenetic analyses.

No MeSH data available.


Related in: MedlinePlus

Group average DNA methylation estimates from pooled and individual DNA samples for the androgen receptor (AR) amplicon on the X-chromosome. Both the pool estimates and group averages based on individual DNA methylation data for the androgen receptor (AR) gene on the X chromosome clearly demonstrate the expected sex differences in DNA methylation with 'offspring' and 'total sample' pools (50% male, 50% female) showing intermediate levels of DNA methylation compared with the high level of methylation in the 'mothers' pool and low level of methylation in the 'fathers' pool.
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Figure 2: Group average DNA methylation estimates from pooled and individual DNA samples for the androgen receptor (AR) amplicon on the X-chromosome. Both the pool estimates and group averages based on individual DNA methylation data for the androgen receptor (AR) gene on the X chromosome clearly demonstrate the expected sex differences in DNA methylation with 'offspring' and 'total sample' pools (50% male, 50% female) showing intermediate levels of DNA methylation compared with the high level of methylation in the 'mothers' pool and low level of methylation in the 'fathers' pool.

Mentions: For those regions located on the X chromosome, the DNA pool results clearly reflected the large sex differences in DNA methylation expected as a result of X-inactivation in females (Figure 2). Furthermore, the pooled DNA accurately estimated group averages across even those regions showing considerable between-individual variation (see Figures 3 and 4). Remarkably, the average absolute difference between the 'pooled' DNA methylation estimate and the 'real' average, determined by assessing individual samples, was 6.0% in the first set of experiments and 4.8% in the second set of replicates. This approximates to the normal level of between-replicate variability expected using the Sequenom EpiTYPER approach [9] and suggests that the accurate pooling of DNA prior to sodium bisulfite treatment does not introduce any significant error beyond that resulting from normal technical variability.


Bisulfite-based epityping on pooled genomic DNA provides an accurate estimate of average group DNA methylation.

Docherty SJ, Davis OS, Haworth CM, Plomin R, Mill J - Epigenetics Chromatin (2009)

Group average DNA methylation estimates from pooled and individual DNA samples for the androgen receptor (AR) amplicon on the X-chromosome. Both the pool estimates and group averages based on individual DNA methylation data for the androgen receptor (AR) gene on the X chromosome clearly demonstrate the expected sex differences in DNA methylation with 'offspring' and 'total sample' pools (50% male, 50% female) showing intermediate levels of DNA methylation compared with the high level of methylation in the 'mothers' pool and low level of methylation in the 'fathers' pool.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657899&req=5

Figure 2: Group average DNA methylation estimates from pooled and individual DNA samples for the androgen receptor (AR) amplicon on the X-chromosome. Both the pool estimates and group averages based on individual DNA methylation data for the androgen receptor (AR) gene on the X chromosome clearly demonstrate the expected sex differences in DNA methylation with 'offspring' and 'total sample' pools (50% male, 50% female) showing intermediate levels of DNA methylation compared with the high level of methylation in the 'mothers' pool and low level of methylation in the 'fathers' pool.
Mentions: For those regions located on the X chromosome, the DNA pool results clearly reflected the large sex differences in DNA methylation expected as a result of X-inactivation in females (Figure 2). Furthermore, the pooled DNA accurately estimated group averages across even those regions showing considerable between-individual variation (see Figures 3 and 4). Remarkably, the average absolute difference between the 'pooled' DNA methylation estimate and the 'real' average, determined by assessing individual samples, was 6.0% in the first set of experiments and 4.8% in the second set of replicates. This approximates to the normal level of between-replicate variability expected using the Sequenom EpiTYPER approach [9] and suggests that the accurate pooling of DNA prior to sodium bisulfite treatment does not introduce any significant error beyond that resulting from normal technical variability.

Bottom Line: Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however, they require relatively large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects.Comparison of data generated from the pooled DNA samples with results averaged across the individual samples comprising each pool revealed highly significant correlations for individual CpG sites across all nine regions, with an average overall correlation across all regions and pools of 0.95 (95% bootstrapped confidence intervals: 0.94 to 0.96).Such an approach can be readily applied to the assessment of disease phenotypes reducing the time, cost and amount of DNA starting material required for large-scale epigenetic analyses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King's College London, De Crespigny Park, Denmark Hill, London, SE5 8AF, UK. spjgsdo@iop.kcl.ac.uk

ABSTRACT

Background: DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however, they require relatively large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. We propose that a high-throughput DNA pooling approach will facilitate the use of emerging methylomic profiling techniques in large samples.

Results: Compared with data generated from 89 individual samples, our analysis of 205 CpG sites spanning nine independent regions of the genome demonstrates that DNA pools can be used to provide an accurate and reliable quantitative estimate of average group DNA methylation. Comparison of data generated from the pooled DNA samples with results averaged across the individual samples comprising each pool revealed highly significant correlations for individual CpG sites across all nine regions, with an average overall correlation across all regions and pools of 0.95 (95% bootstrapped confidence intervals: 0.94 to 0.96).

Conclusion: In this study we demonstrate the validity of using pooled DNA samples to accurately assess group DNA methylation averages. Such an approach can be readily applied to the assessment of disease phenotypes reducing the time, cost and amount of DNA starting material required for large-scale epigenetic analyses.

No MeSH data available.


Related in: MedlinePlus