Digital Restriction Enzyme Analysis of Methylation (DREAM) of HL60 genomic DNA. Homo sapiens

We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Overall design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of HL60 genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.

本研究通过SmaI与XmaI酶分步限制性酶切，对CCCGGG靶位点的5-甲基胞嘧啶（5-methyl cytosine）水平进行分析；将Illumina接头连接至限制性酶切片段，并通过Illumina双端高通量测序读取限制性片段末端的甲基化特异性特征序列。
整体实验设计：采用数字限制性酶切甲基化分析（Digital Restriction Enzyme Analysis of Methylation, DREAM）技术，测定HL60基因组DNA的甲基化谱。向基因组DNA中掺入未甲基化、半甲基化及完全甲基化的标准品，随后使用甲基化敏感酶SmaI（产生平末端）及其耐受甲基化的同裂酶XmaI（产生5'CCGG粘性末端）在CCCGGG位点进行分步酶切，得到可反映CCCGGG位点甲基化状态的不同末端序列。通过Illumina高通量测序对这些末端序列进行分析。将测序读段比对至人类基因组后，通过统计测序读段起始处带有CCGG特征的甲基化读段与带有GGG特征的未甲基化读段的数量，即可确定全基因组范围内单个CCCGGG位点的甲基化状态。