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

BMC Genomics

Methodology Article

Jean-François Théroux¹  Jeffrey A. Gross¹  Carl Ernst¹  Gustavo Turecki¹  Naguib Mechawar¹  Gary G. Chen¹  Alexandre Bramoulle¹  Kathryn Vaillancourt¹  Pierre-Eric Lutz¹  Gilles Maussion¹  Pierre Lepage²  Aurélie Masurel²  Geneviève Bourret²  Ulrike Ehlert³  Elena S. Gardini³ 

[1] Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, H4H 1R3, Verdun, QC, Canada;Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada;University of Zurich, Clinical Psychology and Psychotherapy, Zurich, Switzerland;

关键词: Targeted bisulfite sequencing;    5-methylcytosine;    5-hydroxymethylcytosine;    Next-generation sequencing;    Customized MiSeq sequencing;    Multiplexed PCR-directed BS-amplicons;    Multiplexed barcoding of target amplicon libraries;   

DOI  :  10.1186/s12864-017-3489-9

 received in 2016-07-06, accepted in 2017-01-11,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundEpigenetic 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.ResultsUsing 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.ConclusionsThis 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.

【 授权许可】

CC BY   
© The Author(s). 2017