Abstract 3003: Targeted bisulfite sequencing of CpG island DNA by solution hybrid selection and next-generation sequencing
Authors: Lee Eun Joon , Pei Lirong , Srivastava Gyan , Kushwaha Garima , Trupti Joshi , Choi Jeong-Hyeon , Wang Xinguo , Xu Dong , Zhang Kun , Shi Huidong 
Published: June 2012

Abstract
Recent studies using next-generation sequencing have generated genome-wide, single-base resolution DNA methylation maps. However, it is still very costly to conduct whole genome shotgun bisulfite sequencing. Currently, few methods enable targeted bisulfite sequencing of CpG islands (CGIs) or other specific regions of interest in a highly flexible and efficient manner. Here we present a novel approach that combines solution-phase hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in targeted CGI and promoter regions. We designed 51,466 single strand DNA oligonucleotides (160-mer) which target 23,441 CGIs and the transcription start sites of 19,369 known genes in the human genome. The synthetic long DNA oligonucleotides were converted into biotinylated RNA probes for solution-phase hybridization capture of target DNA. The captured genomic DNA was treated with sodium bisulfite, amplified by PCR and sequenced using Illumina GA IIx sequencer. Using this approach, we conducted bisulfite sequencing on captured DNA from three breast cancer cell lines, MCF10A, MCF7, and MDA-MB-231. 20-30 million single-end 75bp sequencing reads were obtained for each cell line. The raw sequencing reads were mapped to in silico bisulfite-converted human genome. The methylation levels for CpG sites covered with at least 10 sequencing reads were extracted, providing accurate quantification on 900,000-1,000,000 CpGs. 77-84% of CpGs analyzed in these samples fell on or near capture probe sequences; 69-75% lay properly on CGIs. One lane of Illumina sequencing data was sufficient to determine the methylation status of over 22,000 CGIs (75% of all annotated CGIs) in the human genome. More than 85% of capture probes successfully yielded quantitative DNA methylation data of targeted regions. We chose 45 candidate loci (760 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. This novel method was further validated by sequencing three DNA methyltransferase (DNMT) knockout cell lines and the parental HCT116 colon cancer cell line. The targeted bisulfite sequencing data shows massive demethylation events in DNMT1 knockout (1KO) and double knockout (DKO) cell lines, but not in DNMT3B knockout (3BKO) and HCT116 cell lines. While the results indicate the critical role of DNMT1 in maintaining global DNA methylation, we have also identified DNMT3B specific demethylation patterns in a small group of genomic loci. In this study, we demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer genome. 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.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2011-3003