From field to museum: Harnessing the power of third generation sequencing to establish a simple and cost-effective multiplex approach for spider taxonomy

分类学在很大程度上得益于DNA条形码技术的发展。条形码通常是基于对短基因片段的分析，因此称为分子条形码，可用于识别分类群。目前使用的条形码方法通常基于单个标记，其分类分辨率往往有限，因为单个基因的进化通常不能反映物种的进化。近年来，高通量测序技术的发展使得DNA条形码方法中利用的基因位点数量大大增加。分子分类学假设现在可以基于在基因组中取样的成百上千个基因座，甚至可以识别年轻的物种对。然而，这种高通量方法大大增加了DNA条形码的必要工作量和成本。对于面临数百个新物种的识别和描述的分类学家来说，这种高通量的协议通常是昂贵和费力的。在这里，我们的目标是开发一种新的DNA条形码协议，以恢复基因组范围内的多位点数据。合适的DNA条形码标记将通过比较基因组学鉴定，通过长距离PCR扩增，然后使用纳米孔技术进行测序。我们的方案的特点是它的简单性和成本效益，每个样品的成本只有几欧元。此外，我们将开发一种多重PCR方案和一种简化的基因富集试验，从博物馆标本的降解DNA中恢复长条形码序列。我们提出的协议允许在不同的分类群中生成数千个信息性条形码数据的碱基对，并且与目前使用的高通量方法相比，成本大大降低。此外，它们的特点是工作流程大大简化，使世界各地的分类学家都可以使用它们，即使是那些资金和研究基础设施有限的人。

Taxonomy has greatly profited from the development of the method of DNA barcoding. Barcoding is usually based on the analysis of short gene fragments, so called molecular barcodes, which can be used to identify taxa. Currently used barcoding approaches are often based on single markers, whose taxonomic resolution is often limited, as the evolution of a single gene usually does not reflect that of a species. The development of high throughput sequencing technology has made it possible to greatly increase the number of utilized gene loci in DNA barcoding approaches in recent years. Molecular taxonomic hypotheses can now be based on hundreds or thousands of loci sampled across the genome, and allow the identification of even young species pairs. However, such high throughput approaches considerably increase the necessary workload and cost for DNA barcoding. For taxonomists who are facing the identification and description of hundreds of new species, such high throughput protocols are often prohibitively expensive and laborious. Here, we aim to develop a novel DNA barcoding protocol to recover genome wide multi locus data. Suitable DNA barcode markers will be identified by comparative genomics, amplified by long range PCR and then sequenced using Nanopore technology. Our protocol is distinguished by its simplicity and cost efficiency at a cost of few Euros per specimen. Moreover, we will develop a multiplex PCR protocol and a simplified gene enrichment assay to recover long barcode sequences from degraded DNA of museum specimens. Our proposed protocols allow the generation of thousands of basepairs of informative barcode data across divergent taxonomic groups and for considerably reduced cost compared to currently used high throughout methods. In addition, they are distinguished by a greatly simplified workflow making them accessible to taxonomists all over the world, even in those with limited funding and research infrastructure.