title: Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing.
abstract: We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21,408 CGIs and more than 15,946 transcriptional regulatory regions. Of the CpGs analyzed, 77-84% fell on or near capture probe sequences; 69-75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5'-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.
authors.affiliation: GHSU Cancer Center, Department of Biochemistry and Molecular Biology, Georgia Health Sciences University, Augusta, GA 30912, USA.
authors.forename: Eun-Joon
authors.initials: EJ
authors.lastname: Lee
authors.forename: Lirong
authors.initials: L
authors.lastname: Pei
authors.forename: Gyan
authors.initials: G
authors.lastname: Srivastava
authors.forename: Trupti
authors.initials: T
authors.lastname: Joshi
authors.forename: Garima
authors.initials: G
authors.lastname: Kushwaha
authors.forename: Jeong-Hyeon
authors.initials: JH
authors.lastname: Choi
authors.forename: Keith D
authors.initials: KD
authors.lastname: Robertson
authors.forename: Xinguo
authors.initials: X
authors.lastname: Wang
authors.forename: John K
authors.initials: JK
authors.lastname: Colbourne
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.forename: Gary P
authors.initials: GP
authors.lastname: Schroth
authors.forename: Dong
authors.initials: D
authors.lastname: Xu
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Huidong
authors.initials: H
authors.lastname: Shi
mesh_terms: D045744:Cell Line, Tumor; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation; D055786:Gene Knockout Techniques; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
chemical_list: D013447:Sulfites; D004248:DNA (Cytosine-5-)-Methyltransferases; C009279:sodium bisulfite