Prosser Sean W J, deWaard Jeremy R, Miller Scott E, Hebert Paul D N
Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada.
National Museum of Natural History, Smithsonian Institution, Washington, DC, USA.
Mol Ecol Resour. 2016 Mar;16(2):487-97. doi: 10.1111/1755-0998.12474. Epub 2015 Oct 26.
Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next-generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century-old type specimens of Lepidoptera by attempting to recover 164-bp and 94-bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories--high (164-bp sequence), medium (94-bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR-based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.
模式标本具有很高的科学重要性，因为它们提供了林奈名称的应用与物理标本之间唯一确定的联系。许多其他个体可能已被鉴定为某一特定物种，但它们与分类单元概念的联系是推断性的。由于模式标本往往有一个多世纪的历史，并且经历了不利于DNA保存的条件，因此在序列恢复方面能否成功一直不确定。本研究通过采用新一代测序（NGS）从少量模板DNA中恢复细胞色素c氧化酶1基因条形码区域的序列，应对了这一挑战。首先，通过尝试通过桑格测序恢复164 bp和94 bp的读数，对1800多个有百年历史的鳞翅目模式标本的DNA质量进行了筛选。该分析允许将每个标本归入三个DNA质量类别之一——高（164 bp序列）、中（94 bp序列）或低（无序列）。随后，通过一种基于PCR的NGS方案对每个类别的10个标本进行了分析，该方案只需要很少的模板DNA。对于这三个DNA类别，它从所有标本中恢复了序列信息，平均读数长度在458 bp到610 bp之间。通过在每次NGS运行中对10个标本进行测序，成本与桑格分析相似。未来每次运行中处理的标本数量增加有望大幅降低成本，从而有可能预见一个未来大多数模式标本都能获得条形码序列的局面。
