Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine.
- Chen GG Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Gross JA Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Lutz PE Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Vaillancourt K Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Maussion G Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Bramoulle A Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Théroux JF Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Gardini ES University of Zurich, Clinical Psychology and Psychotherapy, Zurich, Switzerland.
- Ehlert U University of Zurich, Clinical Psychology and Psychotherapy, Zurich, Switzerland.
- Bourret G Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada.
- Masurel A Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada.
- Lepage P Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada.
- Mechawar N Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Turecki G Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
- Ernst C 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.
- 2017-01-20
Published in:
- BMC genomics. - 2017
5-hydroxymethylcytosine
5-methylcytosine
Customized MiSeq sequencing
Multiplexed PCR-directed BS-amplicons
Multiplexed barcoding of target amplicon libraries
Next-generation sequencing
Targeted bisulfite sequencing
5-Methylcytosine
Adult
DNA Methylation
Humans
Male
Oxidation-Reduction
Sequence Analysis, DNA
Sulfites
English
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.
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.
- Language
-
- English
- Open access status
- gold
- Identifiers
-
- DOI 10.1186/s12864-017-3489-9
- PMID 28100169
- Persistent URL
- https://sonar.ch/global/documents/47415
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