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

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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.