Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation

Dustin R. Masser, David R. Stanford, Niran Hadad, Cory B. Giles, Jonathan D. Wren, William E. Sonntag, Arlan Richardson, Willard M. Freeman

Research output: Contribution to journalArticle

9 Scopus citations

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.

Original languageEnglish (US)
Article number49
JournalAGE
Volume38
Issue number3
DOIs
StatePublished - Jun 1 2016

    Fingerprint

Keywords

  • Bisulfite sequencing
  • DNA methylation
  • Rat

ASJC Scopus subject areas

  • Aging
  • Geriatrics and Gerontology

Cite this

Masser, D. R., Stanford, D. R., Hadad, N., Giles, C. B., Wren, J. D., Sonntag, W. E., Richardson, A., & Freeman, W. M. (2016). Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation. AGE, 38(3), [49]. https://doi.org/10.1007/s11357-016-9914-1