Darbeheshti Farzaneh, Zeggar Hayet Radia, Salmani Hamzeh, Liu Yibin, Liu Ruolin, Adalsteinsson Viktor A, Makrigiorgos G Mike
bioRxiv. 2025 Jun 30:2025.06.29.660377. doi: 10.1101/2025.06.29.660377.
The epigenetic deregulation of CpG islands (CGIs) plays a crucial role in cancer initiation and progression. CGIs comprise 1-2% of the human genome and are rich in differentially methylated regions (DMRs) that can serve as cancer biomarkers in clinical samples and liquid biopsies. Focusing epigenetic sequencing on CpG-rich sequences, including CGIs and avoiding non-informative regions, offers an efficient and sensitive approach for cancer identification and tracking, especially within samples containing excess of unaltered, normal DNA. To this end, we have developed Adaptor-anchored Methylation amplification via Proximity Primers (aMAPP), a versatile PCR-based enrichment method. aMAPP employs specially designed primers to selectively enrich either methylated or unmethylated CpGs, depending on the upstream methylation conversion method employed. aMAPP achieves high coverage of genome-wide CGIs and detects hundreds of DMRs in tumor samples compared to adjacent normal tissue using ultra-low depth sequencing (∼300,000 reads). It enables tracing of aberrant methylation down to allelic frequency 0.01% in dilutions of tumor DNA and in cell-free DNA samples, can be applied using picogram amounts of DNA, and can be adapted to enrich either small panels of cancer-specific DMRs, or the majority (>90%) of genomic CGIs and CpGs. aMAPP offers a simple, cost-effective, and highly sensitive approach for capturing the epigenetic footprint of genome-wide CpGs and identifying aberrantly methylated or un-methylated genomic regions.
CpG岛（CGIs）的表观遗传失调在癌症的发生和发展中起着至关重要的作用。CGIs占人类基因组的1-2%，富含差异甲基化区域（DMRs），这些区域可作为临床样本和液体活检中的癌症生物标志物。将表观遗传测序聚焦于富含CpG的序列，包括CGIs，并避免非信息区域，为癌症的识别和追踪提供了一种高效且灵敏的方法，尤其是在含有过量未改变的正常DNA的样本中。为此，我们开发了通过邻近引物进行衔接子锚定甲基化扩增（aMAPP），这是一种基于PCR的通用富集方法。aMAPP根据所采用的上游甲基化转化方法，使用专门设计的引物选择性富集甲基化或未甲基化的CpG。与相邻正常组织相比，aMAPP通过超低深度测序（约300,000条 reads）实现了全基因组CGIs的高覆盖，并在肿瘤样本中检测到数百个DMRs。它能够在肿瘤DNA稀释液和游离DNA样本中追踪异常甲基化至等位基因频率0.01%，可以使用皮克量的DNA进行应用，并且可以适用于富集一小部分癌症特异性DMRs，或基因组中大部分（>90%）的CGIs和CpGs。aMAPP为捕获全基因组CpG的表观遗传足迹以及识别异常甲基化或未甲基化的基因组区域提供了一种简单、经济高效且高度灵敏的方法。
