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

PREMISE: Genomic analysis of population structure is important for understanding plant genetic diversity among and within populations. A limitation to the broader application of genetics in conservation and related fields 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 0.5× and 0.1× 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 0.1× reduces costs and generally performs comparably to 0.5× 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, 0.1× libraries had equal or improved performance with no significant decrease in sequencing efficiency. Differences by tissue type likely reflected sampling variation. DISCUSSION: Miniaturization to 0.1× 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.