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

24Citations
Citations of this article
97Readers
Mendeley users who have this article in their library.

Abstract

© 2015 Yang et al.; licensee BioMed Central. 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.

Cite

CITATION STYLE

APA

Yang, Y., Sebra, R., Pullman, B. S., Qiao, W., Peter, I., Desnick, R. J., … Scott, S. A. (2015). Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS). BMC Genomics, 16(1). https://doi.org/10.1186/s12864-015-1572-7

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free