We present a protocol to prepare extracted DNA for sequencing on the Illumina sequencing platform that has been optimized for ancient and degraded DNA. Our approach, the Santa Cruz Reaction or SCR, uses directional splinted ligation of Illumina’s P5 and P7 adapters to convert natively single-stranded DNA and heat denatured double-stranded DNA into sequencing libraries in a single enzymatic reaction. To demonstrate its efficacy in converting degraded DNA molecules, we prepare 5 ancient DNA extracts into sequencing libraries using the SCR and 2 of the most commonly used approaches for preparing degraded DNA for sequencing: BEST, which targets and converts double-stranded DNA, and ssDNA2.0, which targets and converts single-stranded DNA. We then compare the efficiency with which each approach recovers unique molecules, or library complexity, given a standard amount of DNA input. We find that the SCR consistently outperforms the BEST protocol in recovering unique molecules and, despite its relative simplicity to perform and low cost per library, has similar performance to ssDNA2.0 across a wide range of DNA inputs. The SCR is a cost- and time-efficient approach that minimizes the loss of unique molecules and makes accessible a taxonomically, geographically, and a temporally broader sample of preserved remains for genomic analysis.

中文翻译：

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一种针对古代 DNA 优化的快速高效的单链基因组文库制备方法

我们提出了一个协议，用于在 Illumina 测序平台上准备提取的 DNA 进行测序，该平台已针对古代和退化的 DNA 进行了优化。我们的方法，Santa Cruz 反应或 SCR，使用 Illumina 的 P5 和 P7 接头的定向夹板连接，在单个酶促反应中将天然单链 DNA 和热变性双链 DNA 转化为测序文库。为了证明其在转化降解 DNA 分子方面的功效，我们使用 SCR 和 2 种最常用的制备降解 DNA 进行测序的方法将 5 种古代 DNA 提取物制备到测序文库中：BEST，靶向和转化双链 DNA，以及 ssDNA2 .0，它靶向并转化单链 D​​NA。然后，我们比较每种方法恢复独特分子或文库复杂性的效率，给定标准量的 DNA 输入。我们发现 SCR 在恢复独特分子方面始终优于 BEST 协议，尽管其执行相对简单且每个库的成本较低，但在广泛的 DNA 输入中具有与 ssDNA2.0 相似的性能。SCR 是一种具有成本效益和时间效率的方法，可最大限度地减少独特分子的损失，并为基因组分析提供分类学、地理上和时间上更广泛的保存遗骸样本。