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 21408 CGIs and more than 15946 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.
| Original language | English (US) |
|---|---|
| Journal | Nucleic Acids Research |
| Volume | 39 |
| Issue number | 19 |
| DOIs | |
| State | Published - Oct 1 2011 |
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ASJC Scopus subject areas
- Genetics
Cite this
Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing. / Lee, Eun Joon; Pei, Lirong; Srivastava, Gyan; Joshi, Trupti; Kushwaha, Garima; Choi, Jeong-Hyeon; Robertson, Keith D.; Wang, Xinguo; Colbourne, John K.; Zhang, Lu; Schroth, Gary P.; Xu, Dong; Zhang, Kun; Shi, Huidong.
In: Nucleic Acids Research, Vol. 39, No. 19, 01.10.2011.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Targeted bisulfite sequencing by solution hybrid selection and massively parallel sequencing
AU - Lee, Eun Joon
AU - Pei, Lirong
AU - Srivastava, Gyan
AU - Joshi, Trupti
AU - Kushwaha, Garima
AU - Choi, Jeong-Hyeon
AU - Robertson, Keith D.
AU - Wang, Xinguo
AU - Colbourne, John K.
AU - Zhang, Lu
AU - Schroth, Gary P.
AU - Xu, Dong
AU - Zhang, Kun
AU - Shi, Huidong
PY - 2011/10/1
Y1 - 2011/10/1
N2 - 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 21408 CGIs and more than 15946 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.
AB - 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 21408 CGIs and more than 15946 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.
UR - http://www.scopus.com/inward/record.url?scp=80455178802&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80455178802&partnerID=8YFLogxK
U2 - 10.1093/nar/gkr598
DO - 10.1093/nar/gkr598
M3 - Article
C2 - 21785137
AN - SCOPUS:80455178802
VL - 39
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 19
ER -