True single-molecule DNA sequencing of a pleistocene horse bone
True single-molecule DNA sequencing of a pleistocene horse bone
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DOI：
10.1101/gr.122747.111
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发表时间：
2011-10-01
期刊：
影响因子：
7
通讯作者：
Willerslev, Eske
中科院分区：
文献类型：
--
作者：
Orlando, Ludovic;Ginolhac, Aurelien;Willerslev, Eske
第二代测序平台给古代DNA领域带来了革命性的变化，为人们提供了获取过去个体和灭绝物种的完整基因组的途径。然而，这些平台依赖于文库构建和扩增步骤，这可能会导致序列不能反映原始DNA模板组成。对于古代DNA来说尤其如此，模板在死后遭受了广泛的破坏。在这里，我们报告了对古代DNA进行的第一次“真正的单分子测序”的结果。我们使用Helicos HeliScope和Illumina GAIIx平台分别从永久冻土保存的更新世马骨中产生了115.9 Mb和76.9Mb的DNA序列。我们发现，在Helicos数据中，来自马的内源DNA序列的百分比高于Illumina数据。这一结果表明，用于生成古代DNA分子测序库的分子生物学工具，如第二代测序所需的那样，在数据中引入了偏见，降低了测序过程的效率，限制了我们充分探索古代DNA提取物的分子复杂性的能力。我们证明，对标准的Helicos DNA模板制备方案进行简单修改后，该样本的马DNA比例进一步增加了三倍。比较现代DNA和古代DNA中的Helicos特异性偏见和序列错误，也发现古代模板3‘端存在广泛的胞嘧啶脱氨基损伤，表明存在3’-序列突出。我们的结果表明，结合目前的第二代和第三代测序方法，可以以前所未有的方式对古基因组进行测序。
Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first "true single molecule sequencing" of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3' ends of ancient templates, indicating the presence of 3'-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second-and third-generation sequencing approaches.