title: Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Gary G
authors.initials: GG
authors.lastname: Chen
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Gross
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Pierre-Eric
authors.initials: PE
authors.lastname: Lutz
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Vaillancourt
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Gilles
authors.initials: G
authors.lastname: Maussion
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Bramoulle
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Théroux
authors.affiliation: University of Zurich, Clinical Psychology and Psychotherapy, Zurich, Switzerland.
authors.forename: Elena S
authors.initials: ES
authors.lastname: Gardini
authors.affiliation: University of Zurich, Clinical Psychology and Psychotherapy, Zurich, Switzerland.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Ehlert
authors.affiliation: Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Bourret
authors.affiliation: Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Masurel
authors.affiliation: Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lepage
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Naguib
authors.initials: N
authors.lastname: Mechawar
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Turecki
authors.affiliation: Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada. carl.ernst@mcgill.ca.
authors.forename: Carl
authors.initials: C
authors.lastname: Ernst
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000328:Adult; D019175:DNA Methylation / Q000187:drug effects; D006801:Humans; D008297:Male; D010084:Oxidation-Reduction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*
chemical_list: D013447:Sulfites; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; C042345:hydrogen sulfite
keywords: 5-hydroxymethylcytosine; 5-methylcytosine; Customized MiSeq sequencing; Multiplexed PCR-directed BS-amplicons; Multiplexed barcoding of target amplicon libraries; Next-generation sequencing; Targeted bisulfite sequencing