<br>DNA 提取过程中的重复子样本揭示 Malaise trap DNA 元条形码的多样性估计增加,Ecology and Evolution

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DNA 提取过程中的重复子样本揭示 Malaise trap DNA 元条形码的多样性估计增加
Ecology and Evolution ( IF 2.3 ) Pub Date : 2022-11-27 , DOI: 10.1002/ece3.9502
Vera M A Zizka ₁ , Matthias F Geiger ₁ , Thomas Hörren ₂ , Ameli Kirse ₁ , Niklas W Noll ₁ , Livia Schäffler ₁ , Alice M Scherges ₁ , Martin Sorg ₂

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随着DNA元条形码在生物多样性评估中的应用不断增加，各种实验室方案已得到优化，其进一步评估是当前研究的主题。大量样品的均质化以及随后从受损组织的子样品中提取 DNA 是元条形码过程的常见第一阶段。这可以使用浸泡在储存固定剂（例如乙醇）中的样品材料（此处称为“湿”处理）或来自干燥的个体（“干”）来进行。然而，仍然不确定在均质化过程中是否能确保试管内 DNA 的完美混合和均匀分布，以及如果在下游元条形码工作流程中仅处理一小部分被破坏的样品，则不完全混合和由此产生的组织成分变化会在多大程度上影响多样性评估。在这里，我们研究了潮湿和干燥条件下的均质化效率，并测试了破坏组织成分的变化如何影响复杂节肢动物样本的多样性评估。我们考虑了 Malaise trap 批量样品的五个时间间隔，并在两种处理中处理了九个不同的均质组织子样品（每个 20 毫克）。结果表明，干燥材料的多样性评估更加一致，但代价是处理时间更长。两种方法都检测到了相当的 OTU 多样性，并在单次组织提取中揭示了相似的分类群组成。随着 DNA 提取过程中组织子样本数量的增加，两种方法的 OTU 多样性都增加，特别是对于夏季获得的高度多样化的样本。在这里，特别是小型和低生物量类群的检测有所增加。因此，元条形码协议中的多个子样本的处理可以成为增强多样性估计和抵消生物多样性评估中的分类偏差的有用程序。然而，该过程会导致更高的成本和时间投入，并且需要根据具体项目的前景考虑在大规模生物多样性评估中的应用，例如在监测计划中的应用。

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Repeated subsamples during DNA extraction reveal increased diversity estimates in DNA metabarcoding of Malaise traps

With increased application of DNA metabarcoding in biodiversity assessment, various laboratory protocols have been optimized, and their further evaluation is subject of current research. Homogenization of bulk samples and subsequent DNA extraction from a subsample of destructed tissue is a common first stage of the metabarcoding process. This can either be conducted using sample material soaked in a storage fixative, e.g., ethanol (here referred to as “wet” treatment) or from dried individuals (“dry”). However, it remains uncertain if perfect mixing and equal distribution of DNA within the tube is ensured during homogenization and to what extent incomplete mixing and resulting variations in tissue composition affect diversity assessments if only a fraction of the destructed sample is processed in the downstream metabarcoding workflow. Here we investigated the efficiency of homogenization under wet and dry conditions and tested how variations in destructed tissue composition might affect diversity assessments of complex arthropod samples. We considered five time intervals of Malaise trap bulk samples and process nine different subsamples of homogenized tissue (20 mg each) in both treatments. Results indicate a more consistent diversity assessment from dried material, but at the cost of a higher processing time. Both approaches detected comparable OTU diversity and revealed similar taxa compositions in a single tissue extraction. With an increased number of tissue subsamples during DNA extraction, OTU diversity increased for both approaches, especially for highly diverse samples obtained during the summer. Here, particularly the detection of small and low-biomass taxa increased. The processing of multiple subsamples in the metabarcoding protocol can therefore be a helpful procedure to enhance diversity estimates and counteract taxonomic bias in biodiversity assessments. However, the process induces higher costs and time effort and the application in large-scale biodiversity assessment, e.g., in monitoring schemes needs to be considered on project-specific prospects.

更新日期：2022-11-29

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