Circulating-cell free DNA pooled samples for epigenome-wide discovery of methylation biomarkers for colorectal cancer screening

We explored the differential methylation patterns found in cfDNA between healthy controls (individuals with no colorectal findings, NCF) and patients with advanced neoplasia (advanced adenomas and colorectal cancer, AA andCRC) using pooled samples, to determine if pooled serum cfDNA samples can be used to search for non-invasive methylation biomarkers, as an affordable and efficient alternative to tissue biopsy. cfDNA was extracted from serum samples and methylation measurements were assessed with the Infinium MethylationEPIC BeadChip. Data was mainly preprocessed and analyzed with R/Bioconductor packages. Two independent pooled samples were constructed for each pathological group (NCF, AA and CRC) using equal amounts of cfDNA from 5 men and 5 women per pool. Genome-wide DNA methylation in the six pooled samples was analyzed with the Infinium MethylationEPIC BeadChip microarray, covering over 850,000 CpG sites across the genome. Two independent pooled samples were constructed for each pathological group (NCF, AA and CRC) using equal amounts of ccfDNA from 5 men and 5 women per pool. Genome-wide DNA methylation in the six pooled samples was analyzed with the Infinium MethylationEPIC BeadChip microarray, covering over 850,000 CpG sites across the genome.

本研究采用混合样本策略，探究健康对照者（无结直肠异常发现者，no colorectal findings, NCF）与进展性肿瘤患者（进展性腺瘤及结直肠癌，advanced adenomas and colorectal cancer, AA和CRC）的循环游离DNA（circulating cell-free DNA, cfDNA）甲基化差异模式，旨在明确混合血清cfDNA样本能否作为一种经济高效的无创替代方案，替代组织活检以筛选无创甲基化生物标志物。研究人员从血清样本中提取cfDNA，采用Infinium MethylationEPIC BeadChip芯片完成甲基化水平检测。数据的预处理与分析主要依托R/Bioconductor工具包开展。本研究为每个病理分组（NCF、AA、CRC）构建两组独立混合样本，每组分池样本均取自5名男性与5名女性的等量cfDNA。针对6份混合样本开展全基因组DNA甲基化分析，采用Infinium MethylationEPIC BeadChip微阵列芯片，该芯片可覆盖全基因组超过85万个CpG位点。本研究为每个病理分组（NCF、AA、CRC）构建两组独立混合样本，每组分池样本均取自5名男性与5名女性的等量循环游离DNA（ccfDNA）。针对6份混合样本开展全基因组DNA甲基化分析，采用Infinium MethylationEPIC BeadChip微阵列芯片，该芯片可覆盖全基因组超过85万个CpG位点。