decemedip: hierarchical Bayesian modeling for cell type deconvolution of immunoprecipitation-based DNA methylome. decemedip: hierarchical Bayesian modeling for cell type deconvolution of immunoprecipitation-based DNA methylome

MeDIP-seq is an enrichment-based DNA methylation profiling technique that measures abundance of methylated DNA. While this technique offers efficiency advantages over direct methylation profiling, it does not provide absolute quantification of DNAm necessary for cell type deconvolution. We introduce decemedip, a Bayesian hierarchical model for cell type deconvolution of methylated sequencing data that leverages reference atlases derived from direct methylation profiling. We demonstrate its accuracy and robustness through simulation studies and validation on cross-platform measurements, and highlight its utility in identifying tissue-specific and cancer-associated methylation signatures using MeDIP-seq profiling of patient-derived xenografts and cell-free DNA. decemedip is available at \url{https://github.com/nshen7/decemedip}. Overall design: The submitted samples were DNA from the LuCaP PDXs extracted using the DNeasy Blood and Tissue Kit (QIAGEN). Genomic DNA was sheared using a Covaris Sonicator E220, and AMPure XP beads (Beckman Coulter) were used to size select 150 to 250 bp DNA fragments. Library preparation was performed using the KAPA HyperPrep Kit (KAPA Biosystems) according to the manufacturer's protocol. We then performed end-repair, A-tailing, and ligation of NEBNext adaptors (NEBNext Multiplex Oligos for Illumina kit, New England BioLabs). Libraries were digested using the USER enzyme (New England BioLabs). Lamda DNA, consisting of unmethylated and in vitro methylated DNA, was added to prepared libraries to achieve a total amount of 100 ng DNA. Methylated and unmethylated Arabidopsis thaliana DNA (Diagenode) was added for quality control. MeDIP was performed using the MagMeDIP Kit (Diagenode anti-5-methylcytosine (5-mC) antibody, specifically the 33D3 clone) following the manufacturer's protocol. Samples were purified using the iPure Kit v2 (Diagenode). Success of the immunoprecipitation was confirmed using qPCR to detect recovery of the spiked-in Arabidopsis thaliana methylated and unmethylated DNA.

甲基化DNA免疫沉淀测序（MeDIP-seq）是一种基于富集的DNA甲基化谱分析技术，用于检测甲基化DNA的丰度。相较于直接甲基化谱分析技术，该方法具备效率优势，但无法提供细胞类型反卷积所需的DNA甲基化绝对定量数据。我们开发了decemedip——一款面向甲基化测序数据的贝叶斯分层模型，可借助直接甲基化谱分析得到的参考图谱实现细胞类型反卷积。我们通过模拟研究与跨平台测量验证，证实了该模型的准确性与鲁棒性；并利用患者来源异种移植瘤（patient-derived xenografts, PDX）和无细胞DNA（cell-free DNA, cfDNA）的MeDIP-seq谱分析，展示了其在识别组织特异性与癌症相关甲基化特征中的应用价值。decemedip的开源地址为：https://github.com/nshen7/decemedip。

实验整体设计如下：本次提交的样本为使用DNeasy血液与组织试剂盒（QIAGEN）提取的LuCaP PDX来源DNA。采用Covaris Sonicator E220超声仪对基因组DNA进行片段化，随后使用AMPure XP磁珠（Beckman Coulter）筛选150~250 bp的DNA片段。参照试剂盒说明书，使用KAPA HyperPrep试剂盒（KAPA Biosystems）完成文库制备。后续依次进行末端修复、加A尾，并连接NEBNext接头（适配Illumina平台的NEBNext Multiplex Oligos试剂盒，New England BioLabs）。使用USER酶（New England BioLabs）对文库进行酶切处理。向构建完成的文库中加入由未甲基化与体外甲基化DNA组成的λDNA，使总DNA量达到100 ng。同时加入拟南芥（Arabidopsis thaliana）甲基化与未甲基化DNA（Diagenode）作为质量控制参照。参照试剂盒说明书，使用MagMeDIP试剂盒（含Diagenode抗5-甲基胞嘧啶（5-methylcytosine, 5-mC）单克隆抗体，具体为33D3克隆）完成MeDIP实验。使用iPure Kit v2试剂盒（Diagenode）对样本进行纯化。通过qPCR检测掺入的拟南芥甲基化与未甲基化DNA的回收率，以验证免疫沉淀实验的成功与否。