Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA
Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA
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DOI：
10.1038/s41596-019-0202-2
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发表时间：
2019-10-01
期刊：
影响因子：
14.8
通讯作者：
De Carvalho, Daniel D.
中科院分区：
文献类型：
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作者：
Shen, Shu Yi;Burgener, Justin M.;De Carvalho, Daniel D.
循环无细胞DNA（cfDNA）包括来自正常和肿瘤组织的释放到血流中的小DNA片段。最近，由于cfDNA中存在组织特异性标志物，cfDNA的甲基化谱分析作为液体活检工具已经变得突出。我们之前已经报道了无细胞甲基化DNA免疫沉淀和高通量测序（cfMeDIP-seq）作为分析血浆cfDNA的DNA甲基化组的灵敏、低输入、成本有效且无亚硫酸氢盐的方法。cfMeDIP-seq是先前公开的MeDIP-seq方案的延伸，并且适于允许具有低输入（范围从1至10 ng）DNA的样品的甲基化组分析，这通过在免疫沉淀之前添加“填充DNA”来实现。该方案不限于血浆cfDNA;它也可以应用于自然剪切且可用性低的其他样本（例如，尿液cfDNA和脑脊液cfDNA），并且可能适用于癌症检测以外的其他应用，包括产前诊断、心脏病学和免疫反应监测。本文提出的方案应使任何标准分子实验室能够在类似于3-4天内从血浆cfDNA生成cfMeDIP-seq文库。
Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in similar to 3-4 d.