Whole genome sequencing of historical specimens from the world's largest fungal collection yields high-quality assemblies.

High-throughput molecular studies of museum specimens (museomics) have great potential in biodiversity research, but fungal historical collections have scarcely been examined, leading to no comprehensive methodological assessments. Here we present a whole genome sequencing (WGS) project conducted at the Fungarium of the Royal Botanic Gardens, Kew. DNA was extracted from 2104 specimens collected between 1770 and 2023, and we found that the specimen age had the smallest effect, while DNA purification and taxonomic identity had the greatest effect on DNA yield. We barcoded 771 specimens, and WGS was conducted on a subset of 394 specimens that were empirically selected for in-depth analysis. We developed an automated assembly pipeline, integrating 16 different approaches. Starting from 220 libraries (excluding lichenised specimens), we produced 3143 assemblies using these approaches and found that there is no universal assembly method that can provide good-quality genomes for all; rather, different approaches should be used depending on the library size and the specimen's age. Producing high-quality genomes from specimens over 100 yr old is possible by using customised DNA extraction protocols and applying a multimethod bioinformatic approach. Whole genomes from historical collections will enrich genomics resources, accelerating biodiversity and evolutionary research, amongst others.