Long-read sequencing and genome assembly of natural history collection samples and challenging specimens

Advances in long-read sequencing technology have shifted a key challenge in biodiversity genomics from DNA sequencing and genome assembly to obtaining suitable samples. Although flash-frozen samples remain the gold standard, they are often unattainable. In contrast, natural history collections worldwide harbor millions of ethanol-preserved samples, but such samples remain largely unutilized for long-read sequencing. Here, we use ethanol-preserved samples containing kilobase-sized DNA to show that amplification-free long-read sequencing can yield contiguous genome assemblies, which we exemplify using two catfish species. For other samples, where amplification-free protocols failed, we show that a modified protocol employing an alternative polymerase can successfully overcome issues related to long-read sequencing and PCR bias. For the maned sloth Bradypus torquatus, we demonstrate that this modified protocol together with long-range HiC data achieves a high-quality chromosome-level assembly of its 3.1 Gb genome, surpassing the previous 500 Mb genome size limit of the previous protocol. Moreover, our modified protocol also improves assembly quality and completeness for various other species that require amplification because of low sequencing performance or very small DNA amounts in tiny millimeter-sized organisms. Together, by highlighting collections as valuable but underexplored sample resources for long-read sequencing and by facilitating genome assemblies of small and challenging organisms, our study advances biodiversity genomics and global efforts to obtain reference genomes of all eukaryotes.