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Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine


Chen, Gary G; Gross, Jeffrey A; Lutz, Pierre-Eric; Vaillancourt, Kathryn; Maussion, Gilles; Bramoulle, Alexandre; Théroux, Jean-François; Gardini, Elena S; Ehlert, Ulrike; Bourret, Geneviève; Masurel, Aurélie; Lepage, Pierre; Mechawar, Naguib; Turecki, Gustavo; Ernst, Carl (2017). Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine. BMC Genomics, 18(1):96.

Abstract

BACKGROUND
Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing.

RESULTS
Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution. Our results demonstrate the ability of this methodology to detect all levels of cytosine modifications at greater than 100× coverage in large sample sets at low cost compared to other targeted methods.

CONCLUSIONS
This approach can be applied to multiple settings, from candidate gene to clinical studies, and is especially useful for validation of differentially methylated or hydroxymethylated regions following whole-genome analyses.

Abstract

BACKGROUND
Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing.

RESULTS
Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution. Our results demonstrate the ability of this methodology to detect all levels of cytosine modifications at greater than 100× coverage in large sample sets at low cost compared to other targeted methods.

CONCLUSIONS
This approach can be applied to multiple settings, from candidate gene to clinical studies, and is especially useful for validation of differentially methylated or hydroxymethylated regions following whole-genome analyses.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:06 Faculty of Arts > Institute of Psychology
Dewey Decimal Classification:150 Psychology
Date:18 January 2017
Deposited On:24 Jan 2017 09:13
Last Modified:13 Aug 2017 00:32
Publisher:BioMed Central
ISSN:1471-2164
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1186/s12864-017-3489-9
PubMed ID:28100169

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Content: Published Version
Language: English
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Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)

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