使用甲基转移酶定向标记和富集对未修饰DNA进行全基因组分析。

IF 4.5 Q1 BIOCHEMICAL RESEARCH METHODS
Luca Tosti, Calum Mould, Imogen Gatehouse, Anthony C Smith, Krystian Ubych, Valentina Miano, Peter W Laird, Jack Kennefick, Robert K Neely
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引用次数: 0

摘要

我们提出了“Active-Seq”(用于体外表观基因组测序的叠氮化物点击标记),这是一种无需碱基转换的技术,可以使用突变的细菌甲基转移酶和合成制备的辅助因子类似物分离含有未修饰CpG位点的DNA。Active-Seq是一个强大的表观基因组分析平台,具有简单而精简的工作流程,与测序文库制备串联进行,并且与低至1 ng的DNA输入量兼容。我们使用模型DNA寡核苷酸建立了Active-Seq性能的基线,并根据金标准全基因组亚硫酸氢盐测序数据进一步验证。我们展示了该平台在组织源性DNA上的强大性能,并在表观基因组的未甲基化、细胞类型特异性标记区域证明了DNA的富集,为该技术在组织反褶积应用中的未来应用奠定了基础。最后,我们将该技术应用于无细胞DNA样本,概述了一种在结直肠癌患者中进行肿瘤知情疾病分析的方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment.

We present "Active-Seq" (azide click tagging for in vitro epigenomic sequencing), a base-conversion-free technology that enables the isolation of DNA containing unmodified CpG sites using a mutated bacterial methyltransferase enzyme and a synthetically prepared cofactor analog. Active-Seq is a robust epigenomic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation and compatible with DNA input quantities as low as 1 ng. We establish a baseline for the performance of Active-Seq using model DNA oligos and further validate it against gold-standard whole-genome bisulfite sequencing data. We show robust performance of the platform across tissue-derived DNA and demonstrate enrichment of DNA at unmethylated, cell-type-specific marker regions of the epigenome, laying the foundation for the future application of this technology in tissue deconvolution applications. Finally, we apply the technology to cell-free DNA samples, outlining an approach for tumor-informed disease profiling in patients with colorectal cancer.

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来源期刊
Cell Reports Methods
Cell Reports Methods Chemistry (General), Biochemistry, Genetics and Molecular Biology (General), Immunology and Microbiology (General)
CiteScore
3.80
自引率
0.00%
发文量
0
审稿时长
111 days
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