Fun‐sized library prep: Miniaturization is a valid method for per‐sample cost reduction in targeted sequencing of angiosperm DNA

ABSTRACT Premise Genomic analysis of population structure is important to the conservation of plant species of concern. A limitation of using genetic information in conservation is the cost of obtaining large datasets. Targeted sequencing and low-volume robotic liquid handlers can reduce library preparation reaction volumes and costs. Methods We used targeted sequencing via Angiosperms353 to obtain data for 768 samples, 18 of which were identical, at 0.5X and 0.1X reaction volumes. We calculated quality and quantity control statistics to compare the effects of tissue age and library reaction volume on sequencing results for on-target nuclear and off-target plastid genes. Results Library miniaturization to 0.1X reduces costs and generally performs comparably to 0.5X libraries. In the full dataset, Tenth-Volume Only libraries showed smaller insert sizes and fewer genes with mapped sequences, but no reduction in mapped reads. In the Overlap Set, 0.1X had equal or improved performance with no significant decrease in sequencing efficiency. Differences by tissue type likely reflected sampling variation. Conclusions Miniaturization to 0.1X substantially reduces per-sample costs while maintaining comparable sequencing quality across fresh and herbarium angiosperm DNA. Overall, miniaturization provides a reliable, cost-effective approach for targeted sequencing, increasing the feasibility of using herbarium collections and enabling broader access to population-level genomic studies.