Stuttgart State Museum of Natural History, Stuttgart, Germany.
Center for Integrative Biodiversity Discovery, Leibniz Institute for Evolution and Biodiversity Science, Museum für Naturkunde, Berlin, Germany.
Mol Ecol Resour. 2024 Apr;24(3):e13922. doi: 10.1111/1755-0998.13922. Epub 2024 Jan 19.
The use of DNA barcoding is well established for specimen identification and large-scale biodiversity discovery, but remains underutilized for time-sensitive applications such as rapid species discovery in field stations, identifying pests, citizen science projects, and authenticating food. The main reason is that existing express barcoding workflows are either too expensive or can only be used in very well-equipped laboratories by highly-trained staff. We here show an alternative workflow combining rapid DNA extraction with HotSHOT, amplicon production with NextGenPCR thermocyclers, and sequencing with low-cost MinION sequencers. We demonstrate the power of the approach by generating 250 barcodes for 285 specimens within 6 h including specimen identification through BLAST. The workflow required only the following major equipment that easily fits onto a lab bench: Thermocycler, NextGenPCR, microplate sealer, Qubit, and MinION. Based on our results, we argue that simplified barcoding workflows for species-level sorting are now faster, more accurate, and sufficiently cost-effective to replace traditional morpho-species sorting in many projects.
DNA 条形码技术在标本鉴定和大规模生物多样性发现方面已经得到了很好的应用，但在一些时间敏感的应用中，如野外站的快速物种发现、害虫鉴定、公民科学项目和食品鉴定等，仍未得到充分利用。主要原因是现有的快速条形码工作流程要么过于昂贵，要么只能由经过高度训练的人员在设备精良的实验室中使用。在这里，我们展示了一种替代工作流程，该流程将快速 DNA 提取与 HotSHOT 相结合，利用 NextGenPCR 热循环仪进行扩增子生产，利用低成本 MinION 测序仪进行测序。我们通过在 6 小时内为 285 个标本生成 250 个条形码来证明该方法的强大功能，包括通过 BLAST 进行标本鉴定。该工作流程仅需要以下主要设备，这些设备很容易放在实验台上：热循环仪、NextGenPCR、微孔板封口机、qubit 和 MinION。基于我们的结果，我们认为简化的物种分类条形码工作流程现在更快、更准确，并且在许多项目中具有足够的成本效益，可以替代传统的形态物种分类。
