Data from: Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades-old dried museum specimens

Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high-throughput laboratory workflows. The strategy uses hemi-nested, degenerate, M13-tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard-compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, ‘collecting in collections’ is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.由于学界普遍认为老旧标本的DNA存在降解限制，自然历史博物馆作为DNA分析材料来源的潜力远未得到充分挖掘。尽管存在极少数例外，绝大多数旨在获取所有物种序列数据的DNA条形码（DNA barcoding）项目，通常仅使用为该研究专门采集的标本，而非博物馆中大量已完成鉴定的馆藏标本——这一局限源于缺乏适配的聚合酶链式反应（PCR）技术。因此，任何能够拓展博物馆标本在DNA分析中应用价值的技术，都具备极高的研究与应用价值。本研究首先针对针插昆虫标本，探究了标本保存时长与PCR扩增子（amplicon）大小对PCR成功率的影响；随后开发了一套PCR引物组与扩增策略，可大幅提升老旧博物馆标本在DNA条形码研究中的利用率。本研究的PCR成功率与现有少数针对同年代标本的已发表研究结果相比表现优异，且该新策略具备易于适配高通量实验室流程的自动化优势。该策略采用半巢式、简并性且带有M13尾的PCR引物，扩增两段重叠的扩增子：每个扩增子需进行两轮PCR反应（即每个DNA样本共需四轮PCR）。第一轮PCR产物将使用内部引物与M13引物进行二次扩增。两段PCR扩增子共同覆盖了鞘翅目（Coleoptera）、鳞翅目（Lepidoptera）、双翅目（Diptera）、半翅目（Hemiptera）、蜻蜓目（Odonata）以及推测可覆盖的其他昆虫的细胞色素c氧化酶亚基I（COI）基因中559 bp的片段。针对保存时长不超过25年的标本，本研究成功获取符合BARCODE标准的序列数据的比例为67%（84份标本中的56份）；针对保存时长不超过55年的标本，该比例为51%（197份标本中的102份）。考虑到野外采集与标本鉴定所需耗费的时间、成本以及专业技术门槛，‘利用馆藏标本进行研究’是一种可行的替代方案，可让研究者充分利用数十年标本馆馆藏工作所积累的鉴定知识。