全基因组亚硫酸氢盐测序 (BS-Seq) 以单碱基分辨率测量胞嘧啶甲基化变化，可用于分析游离 DNA (cfDNA)。在血浆中，已鉴定出超短单链 cfDNA（uscfDNA，∼50 nt）和 167 bp 双链单核小体无细胞 DNA（mncfDNA）。然而，uscfDNA 的甲基化谱尚未描述。传统的 BS-Seq 工作流程可能没有帮助，因为亚硫酸氢盐转化会将较大的 DNA 降解为较小的片段，导致错误分类为 uscfDNA。我们描述了“5mCAdpBS-Seq”工作流程，其中预甲基化的 5mC（5-甲基胞嘧啶）单链接头在亚硫酸氢盐转化之前与热变性的 cfDNA 连接。该方法仅保留未因亚硫酸氢盐处理而改变的 DNA 片段，从而减少 uscfDNA 甲基化分析的偏差。使用 5mCAdpBS-Seq，与 mncfDNA 相比，uscfDNA 的 DNA 甲基化水平较低 (∼15%)，并且启动子和 CpG 岛富集。低甲基化的 uscfDNA 片段在上游转录起始位点 (TSS) 中富集，富集的强度与造血细胞的表达基因相关。使用组织源解卷积，我们推断 uscfDNA 主要来源于嗜酸性粒细胞、中性粒细胞和单核细胞。作为原理验证，我们证明 uscfDNA 甲基化谱的特征可以区分非小细胞肺癌和非癌症样本。建议将 5mCAdpBS-Seq 工作流程用于任何基于 cfDNA 甲基化的研究。© 作者 2024。由牛津大学出版社代表 Nucleic Acids Research 出版。

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.