Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients. Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients

Genome-wide analysis of cell-free DNA (cfDNA) methylation profile has been recognized as a promising approach for sensitive and specific detection of many cancers. However, scaling such genome-wide assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We have shown that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation with high sensitivity and quantitative tracking of colorectal cancer patient’s response to treatment, at much reduced sequencing cost suitable for frequent monitoring. We also show, for the first time, tumor epigenetic subtyping from cfDNA using Heatrich-BS, which could enable patient stratification from non-invasive liquid biopsy. As such, Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy. Overall design: Analysis of GC content, CpG enrichment and methylation status in K562 genomic DNA

无细胞DNA（cell-free DNA, cfDNA）甲基化图谱的全基因组分析，已被公认为实现多种癌症灵敏且特异检测的极具前景的手段。然而，由于全基因组亚硫酸氢盐测序成本高昂，将这类全基因组检测技术推广至临床转化并不具备可行性。我们的研究证实，占基因组比例较小的富GC区域不仅CpG位点高度富集，还承载了绝大多数癌症特异性甲基化特征。在此，我们报道了一款简便高效的富CpG区域热富集亚硫酸氢盐测序（Heat enrichment of CpG-rich regions for Bisulfite Sequencing, Heatrich-BS）平台，该平台可针对这些极具信息价值的区域开展靶向甲基化图谱分析。我们的新型方法与生物信息学算法，能够以高灵敏度实现精准的肿瘤负荷估算，并对结直肠癌患者的治疗响应进行定量追踪；同时测序成本大幅降低，适用于频繁的临床监测。此外，我们首次证实了利用Heatrich-BS平台从cfDNA中实现肿瘤表观遗传分型的可行性，该技术可通过无创液体活检实现患者分层。因此，Heatrich-BS在基于液体活检的癌症大规模筛查与常规监测领域具备巨大应用潜力。整体实验设计：对K562基因组DNA中的GC含量、CpG富集度及甲基化状态进行分析。