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Natural history collections are the physical repositories of our knowledge on species, the entities of biodiversity. Making this knowledge accessible to society – through, for example, digitisation or the construction of a validated, global DNA barcode library – is of crucial importance. To this end, we developed and streamlined a workflow for ‘museum harvesting’ of authoritatively identified Diptera specimens from the Smithsonian Institution’s National Museum of Natural History. Our detailed workflow includes both on-site and off-site processing through specimen selection, labelling, imaging, tissue sampling, databasing and DNA barcoding. This approach was tested by harvesting and DNA barcoding 941 voucher specimens, representing 32 families, 819 genera and 695 identified species collected from 100 countries. We recovered 867 sequences (> 0 base pairs) with a sequencing success of 88.8% (727 of 819 sequenced genera gained a barcode > 300 base pairs). While Sanger-based methods were more effective for recently-collected specimens, the methods employing next-generation sequencing recovered barcodes for specimens over a century old. The utility of the newly-generated reference barcodes is demonstrated by the subsequent taxonomic assignment of nearly 5000 specimen records in the Barcode of Life Data Systems.

The use of DNA barcoding has revolutionized biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian’s National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximize sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,888 genera and 202 families. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximize output and minimize cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.