The smallest size fractions of plankton, nano- and pico-plankton, have been highlighted due to they accomplish key functions in marine ecosystems. However, the knowledge about some of them is scarce because they are difficult or impossible to be detected and identified with non-DNA-based methodologies. Here we have evaluated five DNA extraction protocols (MT1–MT5) and seven bioinformatic pipelines (P1–P7) to find the best protocol for detecting most of the eukaryotic species of nano- and pico-plankton present in an environmental sample using Ion Torrent technology. The protocol MT3 was the most reproducible methodology, showing less variation among samples, good DNA quality and sufficient quantity to amplify and sequence the eukaryote species, offering the best results after sequencing. For bioinformatic analyses, P1 and P7 resulted in the highest percentage of detection for the difficult-to-detect species in mock communities. However, only P1 avoided the confusion with other closed species during the taxonomic assignment. The final protocols, MT3-P1 (free) and MT3-P7 (private), showed good and consistent results when they were applied to an environmental sample, being a valuable tool to study the eukaryotes present in environmental samples of nano- and pico-plankton, even for the genera that are difficult to be detected by other techniques.

中文翻译：

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用于海洋纳米和微型真核浮游生物分析的 DNA 提取方法和生物信息学管道的评估

浮游生物的最小尺寸部分，纳米和微型浮游生物，由于它们在海洋生态系统中完成关键功能而受到重视。然而，关于它们中的一些的知识很少，因为它们很难或不可能被非基于 DNA 的方法检测和识别。在这里，我们评估了五种 DNA 提取方案 (MT1-MT5) 和七种生物信息学管道 (P1-P7)，以找到使用 Ion Torrent 技术检测环境样本中存在的大多数真核生物纳米和微型浮游生物的最佳方案. MT3 协议是最可重复的方法，显示样本之间的差异较小，DNA 质量良好，数量充足，可扩增和测序真核生物物种，测序后可提供最佳结果。对于生物信息学分析，P1 和 P7 导致模拟社区中难以检测的物种的检测百分比最高。然而，只有 P1 在分类分配过程中避免了与其他封闭物种的混淆。最终的协议 MT3-P1（免费）和 MT3-P7（私有）在应用于环境样品时显示出良好且一致的结果，是研究纳米级和微微级环境样品中存在的真核生物的宝贵工具。浮游生物，即使是其他技术难以检测的属。