PMID- 25943404
OWN - NLM
STAT- MEDLINE
DCOM- 20160115
LR  - 20181113
IS  - 1471-2164 (Electronic)
IS  - 1471-2164 (Linking)
VI  - 16
IP  - 1
DP  - 2015 May 6
TI  - Quantitative and multiplexed DNA methylation analysis using long-read 
      single-molecule real-time bisulfite sequencing (SMRT-BS).
PG  - 350
LID - 10.1186/s12864-015-1572-7 [doi]
LID - 350
AB  - BACKGROUND: DNA methylation has essential roles in transcriptional regulation, 
      imprinting, X chromosome inactivation and other cellular processes, and aberrant 
      CpG methylation is directly involved in the pathogenesis of human imprinting 
      disorders and many cancers. To address the need for a quantitative and highly 
      multiplexed bisulfite sequencing method with long read lengths for targeted CpG 
      methylation analysis, we developed single-molecule real-time bisulfite sequencing 
      (SMRT-BS). RESULTS: Optimized bisulfite conversion and PCR conditions enabled the 
      amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625-1491 
      bp amplicons to SMRT-BS indicated high reproducibility across all amplicon 
      lengths (r=0.972) and low standard deviations (</=0.10) between individual CpG 
      sites sequenced in triplicate. Higher variability in CpG methylation quantitation 
      was correlated with reduced sequencing depth, particularly for intermediately 
      methylated regions. SMRT-BS was validated by orthogonal bisulfite-based 
      microarray (r=0.906; 42 CpG sites) and second generation sequencing (r=0.933; 174 
      CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very 
      acceptable, correlation with both orthogonal methods (r=0.836-0.897 and 
      r=0.892-0.927, respectively) compared to amplicons less than ~1.0 kb 
      (r=0.940-0.951 and r=0.948-0.963, respectively). Multiplexing utility was 
      assessed by simultaneously subjecting four distinct CpG island amplicons (702-866 
      bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average 
      depth of 110X), which identified a spectrum of highly quantitative methylation 
      levels across all interrogated CpG sites and cell lines. CONCLUSIONS: SMRT-BS is 
      a novel, accurate and cost-effective targeted CpG methylation method that is 
      amenable to a high degree of multiplexing with minimal clonal PCR artifacts. 
      Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 
      kb) and the previously reported bisulfite sequencing PCR bias towards 
      unmethylated DNA should be considered when measuring intermediately methylated 
      regions. Coupled with an optimized bisulfite PCR protocol, SMRT-BS is capable of 
      interrogating ~1.5 kb amplicons, which theoretically can cover ~91% of CpG 
      islands in the human genome.
FAU - Yang, Yao
AU  - Yang Y
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. yao.yang@mssm.edu.
FAU - Sebra, Robert
AU  - Sebra R
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. robert.sebra@mssm.edu.
AD  - Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at 
      Mount Sinai, New York, NY, 10029, USA. robert.sebra@mssm.edu.
FAU - Pullman, Benjamin S
AU  - Pullman BS
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. bp2@icloud.com.
FAU - Qiao, Wanqiong
AU  - Qiao W
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. wanqiong.qiao@mssm.edu.
FAU - Peter, Inga
AU  - Peter I
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. inga.peter@mssm.edu.
FAU - Desnick, Robert J
AU  - Desnick RJ
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. robert.desnick@mssm.edu.
FAU - Geyer, C Ronald
AU  - Geyer CR
AD  - Cancer Stem Cell Research Group, University of Saskatchewan, Saskatoon, SK, S7N 
      4H4, Canada. clg595@mail.usask.ca.
FAU - DeCoteau, John F
AU  - DeCoteau JF
AD  - Cancer Stem Cell Research Group, University of Saskatchewan, Saskatoon, SK, S7N 
      4H4, Canada. john.decoteau@usask.ca.
FAU - Scott, Stuart A
AU  - Scott SA
AD  - Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount 
      Sinai, New York, NY, 10029, USA. stuart.scott@mssm.edu.
LA  - eng
SI  - SRA/SRX977540
SI  - SRA/SRX977650
GR  - K23 GM104401/GM/NIGMS NIH HHS/United States
PT  - Journal Article
DEP - 20150506
PL  - England
TA  - BMC Genomics
JT  - BMC genomics
JID - 100965258
RN  - 0 (Sulfites)
RN  - OJ9787WBLU (hydrogen sulfite)
SB  - IM
MH  - Cell Line, Tumor
MH  - DNA Methylation/*drug effects
MH  - Genome, Human/genetics
MH  - Humans
MH  - Polymerase Chain Reaction
MH  - Sequence Analysis, DNA/*methods
MH  - Sulfites/*pharmacology
MH  - Time Factors
PMC - PMC4422326
EDAT- 2015/05/07 06:00
MHDA- 2016/01/16 06:00
PMCR- 2015/05/06
CRDT- 2015/05/07 06:00
PHST- 2014/11/17 00:00 [received]
PHST- 2015/04/23 00:00 [accepted]
PHST- 2015/05/07 06:00 [entrez]
PHST- 2015/05/07 06:00 [pubmed]
PHST- 2016/01/16 06:00 [medline]
PHST- 2015/05/06 00:00 [pmc-release]
AID - 10.1186/s12864-015-1572-7 [pii]
AID - 1572 [pii]
AID - 10.1186/s12864-015-1572-7 [doi]
PST - epublish
SO  - BMC Genomics. 2015 May 6;16(1):350. doi: 10.1186/s12864-015-1572-7.