Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine. (December 2017)

• Record Type: Journal Article
• Authors: Chen, Gary; Gross, Jeffrey; Lutz, Pierre-Eric; Vaillancourt, Kathryn; Maussion, Gilles; Bramoulle, Alexandre; Théroux, Jean-François; Gardini, Elena; Ehlert, Ulrike; Bourret, Geneviève; Masurel, Aurélie; Lepage, Pierre; Mechawar, Naguib; Turecki, Gustavo; Ernst, Carl
• Is Part Of: BMC genomics. Volume 18:Number 1(2017)
• Page Start: 1
• Page End: 12
• Publication Date: 2017-12
• Subjects: Targeted bisulfite sequencing -- 5-methylcytosine -- 5-hydroxymethylcytosine -- Next-generation sequencing -- Customized MiSeq sequencing -- Multiplexed PCR-directed BS-amplicons -- Multiplexed barcoding of target amplicon libraries; Genomes -- Periodicals; Gene mapping -- Periodicals; Genomics -- Periodicals; Base Sequence -- Periodicals; Chromosome Mapping -- Periodicals; Genetic Techniques -- Periodicals; Sequence Analysis, DNA -- Periodicals; 572.8605
• DOI: 10.1186/s12864-017-3489-9
• Languages: English
• ISSNs: 1471-2164

Abstract

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.