Genome-wide maps of H3K27me3 in endpoint Ezh2+/+ or Ezh2-/- Tet ON PyVmT mouse mammary gland tumours

We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in endpoint tumours from Ezh2+/+ or Ezh2-/- Tet ON PyVmT mammary gland tumours. In order to understand the H3K27me3 targets in the context of mouse mamamry gland tumorigenesis, data were generated by deep sequencing a pool of 5 tumours per genoype, in duplicate using Illumina HiSeq2500. Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10). Raw reads were trimmed for length (n>=32), quality (phred score >= 30) and adaptor sequence using fastx v0.0.13.1.Trimmed reads were (pools of 5 different tumors per genotype) then aligned to the mouse reference genome mm10 using BWA v0.5.9 Broad peaks were called using MACS v1.4.1 software (mfold=10,30; bandwith=300; pvalue cutoff=1E-5) using sequenced libraries of input DNA as control. Peak list intersections were done using BEDTools v2.12.0. Binding peaks were considered overlapping if at least 1 base of the peaks overlapped. Our study demonstrated the first mapping of H3K27me3 in the endpint tumours of tetracycline ducible PyVmT mouse mamary tumours. Overall design: Examination of a histone modification in two different genotypes of mouse mammary gland tumours

本研究报道了基于单分子测序技术，对Ezh2野生型（Ezh2+/+）或Ezh2敲除型（Ezh2-/-）的Tet ON型PyVmT乳腺终末期肿瘤的组蛋白修饰开展高通量分析的应用。为阐明小鼠乳腺肿瘤发生过程中组蛋白H3赖氨酸27三甲基化（H3K27me3）的靶标区域，本研究通过Illumina HiSeq2500平台，对每个基因型的5个肿瘤混合样本进行双份深度测序以生成相关数据。通过优化后的数据分析流程，我们将每个样本约3000万条测序读段比对至小鼠参考基因组（版本mm10）。使用fastx v0.0.13.1工具对原始测序读段进行质控修剪：过滤长度≥32bp、Phred质量值≥30的序列，并去除接头序列。修剪后的测序读段（每个基因型对应5个不同肿瘤的混合样本）随后使用BWA v0.5.9比对至小鼠参考基因组mm10。以输入DNA的测序文库作为对照，使用MACS v1.4.1软件（参数设置：mfold=10,30；带宽=300；p值阈值=1E-5）进行宽峰呼叫。使用BEDTools v2.12.0进行峰列表的交集分析；若两个结合峰存在至少1个碱基的重叠区域，则判定为重叠峰。本研究首次完成了四环素诱导型PyVmT小鼠乳腺肿瘤终末期组织中H3K27me3的全基因组定位分析。实验整体设计：对两种不同基因型的小鼠乳腺肿瘤中的组蛋白修饰进行检测。