Genomic analysis of hundreds of individuals is increasingly becoming standard in evolutionary and ecological research. Individual‐based sequencing generates large amounts of valuable data from experimental and field studies, while using preserved samples is an invaluable resource for studying biodiversity in remote areas or across time. Yet, small‐bodied individuals or specimens from collections are often of limited use for genomic analyses due to a lack of suitable extraction and library preparation protocols for preserved or small amounts of tissues. Currently, high‐throughput sequencing in zooplankton is mostly restricted to clonal species, that can be maintained in live cultures to obtain sufficient amounts of tissue, or relies on a whole‐genome amplification step that comes with several biases and high costs. Here, we present a workflow for high‐throughput sequencing of single small individuals omitting the need for prior whole‐genome amplification or live cultures. We establish and demonstrate this method using 27 species of the genus Daphnia, aquatic keystone organisms, and validate it with small‐bodied ostracods. Our workflow is applicable to both live and preserved samples at low costs per sample. We first show that a silica‐column based DNA extraction method resulted in the highest DNA yields for nonpreserved samples while a precipitation‐based technique gave the highest yield for ethanol‐preserved samples and provided the longest DNA fragments. We then successfully performed short‐read whole genome sequencing from single Daphnia specimens and ostracods. Moreover, we assembled a draft reference genome from a single Daphnia individual (>50× coverage) highlighting the value of the workflow for non‐model organisms.

中文翻译：

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优化且经济实惠的高通量测序工作流程，适用于保存和未保存的小型浮游动物标本。

数百人的基因组分析正日益成为进化和生态研究的标准。基于个体的测序从实验和实地研究中产生大量有价值的数据，而使用保存的样本是研究偏远地区或跨时间生物多样性的宝贵资源。然而，由于缺乏用于保存或少量组织的合适提取和文库制备方案，体型较小的个体或收集的标本通常用于基因组分析。目前，浮游动物的高通量测序主要限于克隆物种，可以在活体培养物中维持以获得足够数量的组织，或者依赖于具有多种偏差和高成本的全基因组扩增步骤。这里，我们提出了一种对单个小个体进行高通量测序的工作流程，无需事先进行全基因组扩增或活体培养。我们使用该属的 27 个物种建立并证明了这种方法水蚤，水生基石生物，并用小体型介形虫对其进行验证。我们的工作流程适用于每个样本的低成本和活样本。我们首先表明，基于硅胶柱的 DNA 提取方法对未保存的样品产生最高的 DNA 产量，而基于沉淀的技术对乙醇保存的样品产生最高的产量并提供最长的 DNA 片段。然后，我们成功地对单个水蚤标本和介形虫进行了短读全基因组测序。此外，我们从单个水蚤个体（> 50 倍覆盖率）中组装了一份参考基因组草案，突出了非模式生物工作流程的价值。