In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

在循环游离 DNA 领域，大多数研究都集中在短 DNA 分子（例如，<500 bp）上。长的无细胞 DNA 分子的存在研究很少。在这项研究中，我们证明了单分子实时测序使我们能够检测和分析来自母体血浆中胎儿和母体来源的大部分长 DNA 分子。这些分子超出了短读长测序技术的大小检测限制。在孕早期、孕中期和孕晚期，超过 500 bp 的母体血浆中长游离 DNA 分子的比例分别为 15.5%、19.8% 和 32.3%。观察到的最长的源自胎儿的血浆 DNA 分子为 23,635 bp。长血浆 DNA 分子显示 A 或 G 5' 片段末端占优势。先兆子痫的妊娠表明母体血浆长 DNA 分子减少，以 G 或 A 结尾的选定 5' 4-mer 末端基序的频率降低，以 T 或 C 结尾的选定基序的频率增加。最后，我们开发了一种方法，采用分析长 DNA 分子上一系列 CpG 位点的甲基化模式来确定其组织来源。这种方法在区分胎儿和母体血浆 DNA 分子方面实现了 0.88 的曲线下面积，从而能够确定胎儿基因组中的母体遗传和重组事件。