ABSTRACT

Methylation signatures in cell-free DNA (cfDNA) have shown great sensitivity and specificity in the characterization of tumour status and classification of tumour types, as well as the response to therapy and recurrence. Currently, most cfDNA methylation studies are based on bisulphite conversion, especially targeted bisulphite sequencing, while enrichment-based methods such as cfMeDIP-seq are beginning to show potential. Here, we report an enrichment-based ultra-low input cfDNA methylation profiling method using methyl-CpG binding proteins capture, termed cfMBD-seq. We optimized the conditions for cfMBD capture by adjusting the amount of MethylCap protein along with using methylated filler DNA. Our data show high correlation between low input cfMBD-seq and standard MBD-seq (>1000 ng input). When compared to cfMEDIP-seq, cfMBD-seq demonstrates higher sequencing data quality with more sequenced reads passed filter and less duplicate rate. cfMBD-seq also outperforms cfMeDIP-seq in the enrichment of CpG islands. This new bisulphite-free ultra-low input methylation profiling technology has great potential in non-invasive and cost-effective cancer detection and classification.

摘要

游离 DNA (cfDNA) 中的甲基化特征在表征肿瘤状态和肿瘤类型分类以及对治疗和复发的反应方面表现出极大的敏感性和特异性。目前，大多数 cfDNA 甲基化研究都基于亚硫酸氢盐转化，尤其是靶向亚硫酸氢盐测序，而 cfMeDIP-seq 等基于富集的方法开始显示出潜力。在这里，我们报告了一种使用甲基-CpG 结合蛋白捕获的基于富集的超低输入 cfDNA 甲基化分析方法，称为 cfMBD-seq。我们通过调整 MethylCap 蛋白的量以及使用甲基化填充物 DNA 来优化 cfMBD 捕获的条件。我们的数据显示低输入 cfMBD-seq 和标准 MBD-seq（>1000 ng 输入）之间的高度相关性。与 cfMEDIP-seq 相比，cfMBD-seq 展示了更高的测序数据质量，更多的测序读数通过过滤器和更少的重复率。cfMBD-seq 在 CpG 岛的富集方面也优于 cfMeDIP-seq。这种新的无亚硫酸氢盐超低输入甲基化分析技术在非侵入性和具有成本效益的癌症检测和分类方面具有巨大潜力。