Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.
Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85 % as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.|Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA.|Harold Hamm Diabetes Center, Oklahoma City, OK, USA.
Dustin R
DR
Masser
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.|Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA.|Harold Hamm Diabetes Center, Oklahoma City, OK, USA.|Oklahoma Nathan Shock Center of Excellence in Basic Biology of Aging, Oklahoma City, OK, USA.
David R
DR
Stanford
Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA.|Oklahoma Center for Neuroscience, Oklahoma City, OK, USA.
Niran
N
Hadad
Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
Cory B
CB
Giles
Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA.|Oklahoma Nathan Shock Center of Excellence in Basic Biology of Aging, Oklahoma City, OK, USA.|Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.|Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Jonathan D
JD
Wren
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.|Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA.|Oklahoma Nathan Shock Center of Excellence in Basic Biology of Aging, Oklahoma City, OK, USA.|Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
William E
WE
Sonntag
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.|Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA.|Oklahoma Nathan Shock Center of Excellence in Basic Biology of Aging, Oklahoma City, OK, USA.|Oklahoma City VA Medical Center, Oklahoma City, OK, USA.|Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Arlan
A
Richardson
Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. wfreeman@ouhsc.edu.|Reynolds Oklahoma Center on Aging, Oklahoma City, OK, USA. wfreeman@ouhsc.edu.|Harold Hamm Diabetes Center, Oklahoma City, OK, USA. wfreeman@ouhsc.edu.|Oklahoma Nathan Shock Center of Excellence in Basic Biology of Aging, Oklahoma City, OK, USA. wfreeman@ouhsc.edu.|Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. wfreeman@ouhsc.edu.|Department of Physiology OUHSC, BSMB 653, P.O. Box 26901, Oklahoma City, OK, 73126, USA. wfreeman@ouhsc.edu.
Willard M
WM
Freeman
D044503:5-Methylcytosine / Q000032:analysis*; D000375:Aging / Q000235:genetics*; D000818:Animals; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000235:genetics*; D051381:Rats; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000032:analysis*
D013447:Sulfites; D044503:5-Methylcytosine; D004247:DNA; C042345:hydrogen sulfite
Bisulfite sequencing; DNA methylation; Rat