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

Autor: Yang Y; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. yao.yang@mssm.edu., Sebra R; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. robert.sebra@mssm.edu.; Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. robert.sebra@mssm.edu., Pullman BS; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. bp2@icloud.com., Qiao W; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. wanqiong.qiao@mssm.edu., Peter I; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. inga.peter@mssm.edu., Desnick RJ; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. robert.desnick@mssm.edu., Geyer CR; Cancer Stem Cell Research Group, University of Saskatchewan, Saskatoon, SK, S7N 4H4, Canada. clg595@mail.usask.ca., DeCoteau JF; Cancer Stem Cell Research Group, University of Saskatchewan, Saskatoon, SK, S7N 4H4, Canada. john.decoteau@usask.ca., Scott SA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. stuart.scott@mssm.edu.
Jazyk: angličtina
Zdroj: BMC genomics [BMC Genomics] 2015 May 06; Vol. 16, pp. 350. Date of Electronic Publication: 2015 May 06.
DOI: 10.1186/s12864-015-1572-7
Abstrakt: 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.
Databáze: MEDLINE
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