Center for Integrative Biodiversity Discovery, Leibniz Institute for Evolution and Biodiversity Science, Museum für Naturkunde, Berlin, Germany.
Institute for Biology, Humboldt University, Berlin, Germany.
Methods Mol Biol. 2024;2744:223-238. doi: 10.1007/978-1-0716-3581-0_14.
DNA barcodes are useful in biodiversity research, but sequencing barcodes with dye termination methods ("Sanger sequencing") has been so time-consuming and expensive that DNA barcodes are not as widely used as they should be. Fortunately, MinION sequencers from Oxford Nanopore Technologies have recently emerged as a cost-effective and efficient alternative for barcoding. MinION barcodes are now suitable for large-scale species discovery and enable specimen identification when the target species are represented in barcode databases. With a MinION, it is possible to obtain 10,000 barcodes from a single flow cell at a cost of less than 0.10 USD per specimen. Additionally, a Flongle flow cell can be used for small projects requiring up to 300 barcodes (0.50 USD per specimen). We here describe a cost-effective laboratory workflow for obtaining tagged amplicons, preparing ONT libraries, sequencing amplicon pools, and analyzing the MinION reads with the software ONTbarcoder. This workflow has been shown to yield highly accurate barcodes that are 99.99% identical to Sanger barcodes. Overall, we propose that the use of MinION for DNA barcoding is an attractive option for all researchers in need of a cost-effective and efficient solution for large-scale species discovery and specimen identification.
DNA 条码在生物多样性研究中很有用，但由于染料终止法（“桑格测序”）测序条码既耗时又昂贵，因此 DNA 条码的应用并不广泛。幸运的是，牛津纳米孔技术公司的 MinION 测序仪最近成为了一种具有成本效益和高效率的条码替代方法。MinION 条码现在适用于大规模的物种发现，并能够在目标物种在条码数据库中有代表时进行标本鉴定。使用 MinION，从单个流动池获得 10000 个条码的成本不到每个标本 0.10 美元。此外，Flongle 流动池可用于需要多达 300 个条码的小型项目（每个标本 0.50 美元）。我们在这里描述了一种具有成本效益的实验室工作流程，用于获得标记的扩增子、制备 ONT 文库、对扩增子池进行测序，并使用 ONTbarcoder 软件分析 MinION 读取结果。该工作流程已被证明能够产生高度准确的条码，与桑格条码的相似度达到 99.99%。总体而言，我们建议使用 MinION 进行 DNA 条码测序是所有需要经济高效和高效解决方案进行大规模物种发现和标本鉴定的研究人员的一个有吸引力的选择。
