Yang Yao, Sebra Robert, Pullman Benjamin S, Qiao Wanqiong, Peter Inga, Desnick Robert J, Geyer C Ronald, DeCoteau John F, Scott Stuart A
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
BMC Genomics. 2015 May 6;16(1):350. doi: 10.1186/s12864-015-1572-7.
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).
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.
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.
DNA甲基化在转录调控、印记、X染色体失活及其他细胞过程中发挥着重要作用，异常的CpG甲基化直接参与人类印记障碍和多种癌症的发病机制。为满足对一种用于靶向CpG甲基化分析的长读长定量且高度多重亚硫酸氢盐测序方法的需求，我们开发了单分子实时亚硫酸氢盐测序（SMRT-BS）。
优化的亚硫酸氢盐转化和PCR条件能够扩增长达约1.5 kb的DNA片段，对重叠的625 - 1491 bp扩增子进行SMRT-BS分析表明，在所有扩增子长度上具有高重复性（r = 0.972），且在一式三份测序的各个CpG位点之间标准差较低（≤0.10）。CpG甲基化定量中较高的变异性与测序深度降低相关，特别是对于中等甲基化区域。SMRT-BS通过基于亚硫酸氢盐的正交微阵列（r = 0.906；42个CpG位点）和第二代测序（r = 0.933；174个CpG位点）进行了验证；然而，与小于约1.0 kb的扩增子（分别为r = 0.940 - 0.951和r = 0.948 - 0.963）相比，较长的SMRT-BS扩增子（>1.0 kb）与两种正交方法的相关性降低，但仍非常可接受（分别为r = 0.836 - 0.897和r = 0.892 - 0.927）。通过同时对四个不同的CpG岛扩增子（702 - 866 bp；325个CpG）和30个血液系统恶性肿瘤细胞系进行SMRT-BS分析（平均深度为110X）来评估多重实用性，该分析在所有检测的CpG位点和细胞系中确定了一系列高度定量的甲基化水平。
SMRT-BS是一种新颖、准确且具有成本效益的靶向CpG甲基化方法，适用于高度多重分析且克隆PCR假象最少。在检测较长扩增子（>1.0 kb）时需要增加测序深度，并且在测量中等甲基化区域时应考虑先前报道的亚硫酸氢盐测序PCR对未甲基化DNA的偏向性。结合优化的亚硫酸氢盐PCR方案，SMRT-BS能够检测约1.5 kb的扩增子，理论上可覆盖人类基因组中约91%的CpG岛。
