Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Nat Protoc. 2019 Oct;14(10):2749-2780. doi: 10.1038/s41596-019-0202-2. Epub 2019 Aug 30.
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 ~3-4 d.
循环无细胞 DNA（cfDNA）由源自正常和肿瘤组织的小 DNA 片段组成，这些片段被释放到血液中。最近，由于 cfDNA 中存在组织特异性标志物，cfDNA 的甲基化分析作为一种液体活检工具越来越受到关注。我们之前报道了无细胞甲基化 DNA 免疫沉淀和高通量测序（cfMeDIP-seq），作为一种敏感、低输入、具有成本效益且无需亚硫酸氢盐的方法，用于分析血浆 cfDNA 的 DNA 甲基化组。cfMeDIP-seq 是之前发表的 MeDIP-seq 方案的扩展，经过改编后可用于对输入量低（1-10ng）的 DNA 样本进行甲基化组分析，这是通过在免疫沉淀前添加“填充 DNA”实现的。该方案不仅限于血浆 cfDNA；它还可以应用于其他天然片段化且可用性低的样本（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。本文提供的方案应能使任何标准分子实验室在 3-4 天内从血浆 cfDNA 中生成 cfMeDIP-seq 文库。
