Marine sedimentary ancient DNA (sed aDNA) provides a powerful means to reconstruct marine palaeo‐communities across the food web. However, currently there are few optimized sed aDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sed aDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead‐beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica‐solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size‐selection of low molecular‐weight (LMW) DNA to increase the extraction efficiency of sed aDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead‐beating, DNA binding in silica‐solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post‐library LMW size‐selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sed aDNA studies. The new extraction and data‐processing protocol should improve quantitative paleo‐monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.

中文翻译：

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从海洋沉积物中提取古代真核生物DNA的优化方法。

海洋沉积古DNA（sed aDNA）提供了一种强大的手段来重建整个食物网中的海洋古群落。但是，目前很少有优化的sed aDNA提取方案可用于在广泛的真核生物中最大化古代DNA（aDNA）的典型小DNA片段的产量。我们比较了sed的七个组合使用2018年在塔斯马尼亚州玛丽亚岛（Maria Island）距水深104 m处收集的海洋沉积物进行aDNA提取处理和测序文库制备。这七种方法将冷冻沉积物与冷藏沉积物进行了对比，通过珠击诱导的细胞裂解与乙二胺四乙酸（EDTA）孵育，硅胶旋转柱中的DNA结合硅胶溶液中的DNA结合，shot弹枪文库制备物中的DNA稀释与未稀释的DNA结合，以测试扩增步骤中的潜在抑制问题，以及选择低分子量（LMW）DNA的大小选择以提高sed的提取效率DNA。在45个海洋真核生物类群中，冷冻沉淀物经过EDTA孵育和磁珠敲打，硅胶溶液中的DNA结合以及shot弹枪库中未稀释的DNA的结合，获得了最大的效率。我们提出了结合这些步骤的优化提取方案，以及可选的库后LMW大小选择步骤，以保留≤500个碱基对的DNA片段。我们还描述了用于宏基因组学数据的严格的生物信息学过滤方法，并提供了完整的污染物清单，以作为未来sed aDNA研究的参考。新的提取和数据处理方案将改善海洋沉积物中真核生物的定量古监测，以及其他依靠检测沉积物中高度破碎和降解的真核生物DNA的研究。