Pyrosequencing for Mini-Barcoding of Fresh and Old Museum Specimens

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（DNA barcoding）是开展物种鉴定以及发现新物种或隐存物种的有效途径。桑格测序（Sanger sequencing）技术是获取标准650碱基对细胞色素c氧化酶亚基I（COI）条形码的首选方法。然而，DNA降解与片段化使得从老旧标本中获取全长条形码颇具难度。源自标准条形码区域的130碱基对微型条形码，已被证实可在众多动物类群中实现精准鉴定，且可从馆藏标本中便捷获取。本研究验证了一种替代测序技术——四酶单标本焦磷酸测序（four-enzymes single-specimen pyrosequencing）在快速、低成本微型条形码分析中的应用效果。我们从135份新鲜标本与50份老旧鳞翅目（Lepidoptera）标本（标本年代跨度为53至97年）的COI微型条形码片段中，成功获得了最长达100碱基对的序列。经焦磷酸测序得到的序列质量优异，我们可将所有经焦磷酸测序的样本与对应的桑格测序标准条形码序列（若有留存）进行可靠匹配。该实验流程与仪器操作简便，且相较于桑格测序，其测序速度更快、单序列成本更低，这使得该方法有望用于将未鉴定标本的标准条形码序列与仅存有短DNA片段的已知馆藏标本进行关联的相关研究工作。