Abstract
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.
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Acknowledgments
The authors wish to thank Dr. Graham Wiley, Dr. Patrick Gaffney, and the Oklahoma Medical Research Foundation Genomics facility for the sequencing services and John Ferguson for aiding in figure generation. This work was supported the Donald W. Reynolds Foundation, the National Institute on Aging [R01AG026607, P30AG050911], National Eye Institute [R01EY021716, T32EY023202], National Institute on Drug Abuse [R21DA029405], Oklahoma Center for Advancement of Science and Technology [HR14-174], and in part by an award from Harold Hamm Diabetes Center at the University of Oklahoma.
Author contributions
D.R.M, D.R.S, and W.M.F designed the study. D.R.M generated the study data. D.R.M, D.R.S, N.H., C.B.G, J.D.W, and W.M.F performed analysis of data. D.R.M, D.R.S, and W.M.F wrote the manuscript. W.E.S and A.R. provided intellectual input and design considerations. All authors reviewed and contributed to the final manuscript.
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The data set supporting the results of this article is available in the NCBI Sequencing Read Archive (http://www.ncbi.nlm.nih.gov/sra) as fastqs under accession number SRX1117302.
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Masser, D.R., Stanford, D.R., Hadad, N. et al. Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation. AGE 38, 49 (2016). https://doi.org/10.1007/s11357-016-9914-1
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DOI: https://doi.org/10.1007/s11357-016-9914-1