Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.
Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.
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DOI：
10.1038/srep27859
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发表时间：
2016-06-14
影响因子：
4.6
通讯作者：
De Vlaminck I
中科院分区：
文献类型：
--
作者：
Burnham P;Kim MS;Agbor-Enoh S;Luikart H;Valantine HA;Khush KK;De Vlaminck I
循环游离 DNA (cfDNA) 正在成为癌症、妊娠和器官移植领域的强大监测工具。核小体 DNA 是血浆 cfDNA 的主要形式，可通过连接双链 DNA (dsDNA) 接头进行测序。然而，dsDNA 文库制备对超短、降解的 cfDNA 不敏感。受到古基因组学进展的启发，我们应用单链 DNA (ssDNA) 文库制备方法对肺移植受者血浆中的 cfDNA 进行测序（40 个样本，6 名患者）。我们发现 ssDNA 文库制备产生了大部分低于 100bp 的核基因组 cfDNA（p 10−5，Mann-Whitney U 检验），并且线粒体（10.7x，p 10−5）和微生物 cfDNA（71.3x，p 10−5）的相对丰度增加。该方法产生的微生物序列产量较高，提高了基于 cfDNA 的移植后感染监测的灵敏度。我们详细介绍了线粒体、核基因组和微生物 cfDNA 在广泛片段长度范围内的片段模式。我们报告了肺移植受者循环中供体特异性线粒体 cfDNA 的观察结果。 ssDNA 文库制备方法为研究中的 cfDNA 形式（包括线粒体和微生物衍生的 cfDNA 以及短核基因组 cfDNA）提供了更多信息的窗口，同时保留了标准 dsDNA 文库制备方法提供的信息。
Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10−5, Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10−5) and microbial cfDNA (71.3x, p 10−5). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods.