Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's r = 0.71 vs. −0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in Dffb-deficient mice but higher in Dnase1l3-deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules.

中文翻译：

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人血浆中长无细胞 DNA 分子的基因组起源、片段组学和转录特性

最近的研究利用长读长测序技术揭示了人类血浆中未经探索的长无细胞 DNA (cfDNA) 分子群体。然而，长 cfDNA 分子（> 500 bp）的生物学特性仍然很大程度上未知。为此，我们研究了来自不同基因组元件的长 cfDNA 分子的起源。使用长读长测序对血浆 cfDNA 进行分析揭示了整个基因组中长分子的分布不均匀。与短 DNA 分子形成鲜明对比的是，长 cfDNA 分子在基因组的常染色质区域中表现出过度表达。与短分子相比，我们观察到长分子的丰度和 mRNA 基因表达水平之间的关系更强（Pearson's r = 0.71 vs. -0.14）。此外，长分子和短分子在 CpG 位点周围显示出不同的断裂模式。利用 CpG 位点周围的裂解偏好，长分子和短分子的组合裂解比率可以将肝细胞癌 (HCC) 患者与非 HCC 受试者区分开来 (AUC = 0.87)。我们还研究了与野生型小鼠相比，选定的核酸酶基因已失活的基因敲除小鼠。与野生型小鼠相比，Dffb缺陷型小鼠中源自转录起始位点的长分子比例较低，但Dnase1l3缺陷型小鼠中源自转录起始位点的长分子比例较高。因此，这项工作为长 cfDNA 分子的生物学特性和潜在临床应用提供了新的见解。