Extensive manipulations involved in the preparation of DNA samples for sequencing have hitherto made it impossible to determine the precise structure of double-stranded DNA fragments being sequenced, such as the presence of blunt ends, single-stranded overhangs, or single-strand breaks. We here describe MatchSeq, a method that combines single-stranded DNA library preparation from diluted DNA samples with computational sequence matching, allowing the reconstruction of double-stranded DNA fragments on a single-molecule level. The application of MatchSeq to Neanderthal DNA, a particularly complex source of degraded DNA, reveals that 1- or 2-nt overhangs and blunt ends dominate the ends of ancient DNA molecules and that short gaps exist, which are predominantly caused by the loss of individual purines. We further show that deamination of cytosine to uracil occurs in both single- and double-stranded contexts close to the ends of molecules, and that single-stranded parts of DNA fragments are enriched in pyrimidines. MatchSeq provides unprecedented resolution for interrogating the structures of fragmented double-stranded DNA and can be applied to fragmented double-stranded DNA isolated from any biological source. The method relies on well-established laboratory techniques and can easily be integrated into routine data generation. This possibility is shown by the successful reconstruction of double-stranded DNA fragments from previously published single-stranded sequence data, allowing a more comprehensive characterization of the biochemical properties not only of ancient DNA but also of cell-free DNA from human blood plasma, a clinically relevant marker for the diagnosis and monitoring of disease.

中文翻译：

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在单分子水平上重建双链 DNA 片段揭示了古代样本中的降解模式。

迄今为止，在制备用于测序的 DNA 样品中涉及的大量操作使得无法确定被测序的双链 DNA 片段的精确结构，例如平末端、单链突出端或单链断裂的存在。我们在此描述 MatchSeq，这是一种将稀释 DNA 样本的单链 DNA 文库制备与计算序列匹配相结合的方法，允许在单分子水平上重建双链 DNA 片段。将 MatchSeq 应用于尼安德特人 DNA，这是一种特别复杂的降解 DNA 来源，揭示了 1 或 2 nt 突出端和平末端主导了古代 DNA 分子的末端，并且存在短间隙，这主要是由个体的缺失引起的。嘌呤。我们进一步表明，在靠近分子末端的单链和双链环境中，胞嘧啶脱氨为尿嘧啶，并且 DNA 片段的单链部分富含嘧啶。MatchSeq 为研究片段化双链 DNA 的结构提供了前所未有的分辨率，并可应用于从任何生物来源分离的片段化双链 DNA。该方法依赖于成熟的实验室技术，可以轻松集成到常规数据生成中。从先前发表的单链序列数据成功重建双链 DNA 片段表明了这种可能性，不仅可以更全面地表征古代 DNA 的生化特性，还可以更全面地表征来自人血浆的无细胞 DNA，