title: Application of microdroplet PCR for large-scale targeted bisulfite sequencing.
abstract: Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: H Kiyomi
authors.initials: HK
authors.lastname: Komori
authors.forename: Sarah A
authors.initials: SA
authors.lastname: LaMere
authors.forename: Ali
authors.initials: A
authors.lastname: Torkamani
authors.forename: G Traver
authors.initials: GT
authors.lastname: Hart
authors.forename: Steve
authors.initials: S
authors.lastname: Kotsopoulos
authors.forename: Jason
authors.initials: J
authors.lastname: Warner
authors.forename: Michael L
authors.initials: ML
authors.lastname: Samuels
authors.forename: Jeff
authors.initials: J
authors.lastname: Olson
authors.forename: Steven R
authors.initials: SR
authors.lastname: Head
authors.forename: Phillip
authors.initials: P
authors.lastname: Ordoukhanian
authors.forename: Pauline L
authors.initials: PL
authors.lastname: Lee
authors.forename: Darren R
authors.initials: DR
authors.lastname: Link
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Salomon
mesh_terms: D001483:Base Sequence; D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D044127:Epigenesis, Genetic; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D008832:Microchemistry / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
chemical_list: D017931:DNA Primers; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite