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.

Essential Role for Dnmt1 in the Prevention and Maintenance of MYC-Induced T-Cell Lymphomas

“…Previous studies have identified targets of Dnmt1-dependent methylation and transcription in the HCT116 colon cancer cell line (59,60). When we compared these target genes with Dnmt1 targets identified in this study, we observed only a 2% overlap (data not shown), suggesting that Dnmt1 may have tissue-or species-specific target loci.…”

DNA Methylome Analyses Implicate Fallopian Tube Epithelia as the Origin for High-Grade Serous Ovarian Cancer

“…We performed Agilent SureSelect Methylome bisulfite sequencing (methyl-seq), a targeted solution hybridization method (32), which analyzed methylation at ~4 × 10 6 CpGs, at the University of Nebraska Medical Center (UNMC) Epigenomics Core (n=7) (Supplementary Table S1). We used either the Zymo Pico Methyl-Seq or Agilent SureSelect Methyl-Seq Kit for library preparations, and Agilent SureSelect baits to pull down the final sequences.…”

Masculine Epigenetic Sex Marks of the CYP19A1/Aromatase Promoter in Genetically Male Chicken Embryonic Gonads Are Resistant to Estrogen-Induced Phenotypic Sex Conversion1

“…Debates are ongoing as to whether such transgenerationally transmitted, diseaserelevant epimutations may require specific genetic backgrounds of the laboratory animals [48] or might not occur as significant threats to human and wildlife health with environmentally relevant concentrations of the epimutagens chemicals [49]. Our present study demonstrates the importance of examining changes in DNA methylation and other epigenetic marks caused by fetal exposure to environmental factors with the single nucleotide resolution power using the latest highthroughput technologies, including base-specific cleavage mass spectrometry [47,50] and bisulfite deep sequencing [51][52][53] in addition to the standard bisulfite pyrosequencing approach. Future studies should address the mechanistic basis of this apparent nucleotide-level specificity of epigenetic effects of embryonic exposure to environmental hormonal agents.…”