Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS)

Yao Yang, Robert Sebra, Benjamin S. Pullman, Wanqiong Qiao, Inga Peter, Robert J. Desnick, C. Ronald Geyer, John F. DeCoteau, Stuart A. Scott

Research output: Contribution to journalArticlepeer-review

62 Scopus citations


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 ~1.5kb, and subjecting overlapping 625-1491bp 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 (>1.0kb) 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 ~1.0kb (r = 0.940-0.951 and r = 0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702-866bp; 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 (>1.0kb) 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 ~1.5kb amplicons, which theoretically can cover ~91% of CpG islands in the human genome.

Original languageEnglish
Article number350
JournalBMC Genomics
Issue number1
StatePublished - 6 May 2015


  • Bisulfite sequencing
  • CpG islands
  • DNA methylation
  • Long-read sequencing
  • Pacific Bioscience
  • Single-molecule real-time (SMRT) sequencing
  • Third generation sequencing


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