Methylated DNA Immunoprecipitation and High-Throughput Sequencing (MeDIP-seq) Using Low Amounts of Genomic DNA

甲基化DNA免疫沉淀 DNA甲基化 生物 差异甲基化区 基因组DNA 5-甲基胞嘧啶 照明菌甲基化试验 基因组印记 遗传学 计算生物学 DNA 分子生物学 基因 基因表达
作者
Zhao Mt,Whyte Jj,Hopkins Gm,Kirk,Randall S. Prather
出处
期刊:Cellular Reprogramming [Mary Ann Liebert, Inc.]
卷期号:16 (3): 175-184 被引量:46
标识
DOI:10.1089/cell.2014.0002
摘要

DNA modifications, such as methylation and hydroxymethylation, are pivotal players in modulating gene expression, genomic imprinting, X-chromosome inactivation, and silencing repetitive sequences during embryonic development. Aberrant DNA modifications lead to embryonic and postnatal abnormalities and serious human diseases, such as cancer. Comprehensive genome-wide DNA methylation and hydroxymethylation studies provide a way to thoroughly understand normal development and to identify potential epigenetic mutations in human diseases. Here we established a working protocol for methylated DNA immunoprecipitation combined with next-generation sequencing [methylated DNA immunoprecipitation (MeDIP)-seq] for low starting amounts of genomic DNA. By using spike-in control DNA sets with standard cytosine, 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC), we demonstrate the preferential binding of antibodies to 5mC and 5hmC, respectively. MeDIP-PCRs successfully targeted highly methylated genomic loci with starting genomic DNA as low as 1 ng. The enrichment efficiency declined for constant spiked-in controls but increased for endogenous methylated regions. A MeDIP-seq library was constructed starting with 1 ng of DNA, with the majority of fragments between 250 bp and 600 bp. The MeDIP-seq reads showed higher quality than the Input control. However, after being preprocessed by Cutadapt, MeDIP (97.53%) and Input (94.98%) reads showed comparable alignment rates. SeqMonk visualization tools indicated MeDIP-seq reads were less uniformly distributed across the genome than Input reads. Several commonly known unmethylated and methylated genomic loci showed consistent methylation patterns in the MeDIP-seq data. Thus, we provide proof-of-principle that MeDIP-seq technology is feasible to profile genome-wide DNA methylation in minute DNA samples, such as oocytes, early embryos, and human biopsies.
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