Biodiversity Institute of Ontario, Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.
PLoS One. 2011;6(7):e21252. doi: 10.1371/journal.pone.0021252. Epub 2011 Jul 27.
DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53-97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments.
DNA 条形码是一种有效的物种鉴定方法，可用于发现新的和/或隐藏的物种。桑格测序技术是获取标准 650bp 细胞色素 c 氧化酶亚基 I（COI）条形码的首选方法。然而，DNA 降解/碎片化使得从旧标本中获得全长条形码变得困难。来自标准条形码区域的 130bp 迷你条形码已被证明在许多动物群体中用于准确鉴定是有效的，并且可以从博物馆样本中轻易获得。在这里，我们展示了一种替代测序技术——四酶单样本焦磷酸测序，在快速、具有成本效益的迷你条形码分析中的应用。我们能够从 135 个新鲜和 50 个陈旧鳞翅目标本（年龄在 53-97 岁之间）的 COI 迷你条形码片段中生成多达 100bp 的序列。使用焦磷酸测序获得的序列质量很高，并且我们能够将所有测试的焦磷酸测序样本与其各自的桑格测序标准条形码序列进行稳健匹配，只要有可用的序列。该方法的协议和仪器简单，与桑格测序相比，每序列的速度更快，成本更低，因此这种方法可能有助于将未知标本的标准条形码序列与只有短 DNA 片段的已知博物馆标本联系起来。
