Abstract
Buccal swabs offer a minimally invasive DNA sampling method for wildlife but are often considered suboptimal for genomic applications due to low and variable DNA yields. This limitation has restricted their use in reduced-representation sequencing approaches, which typically require relatively high DNA input mass. We evaluated the utility of buccal swabs collected from Red-cockaded Woodpecker ( Dryobates borealis ) nestlings as a source of DNA for genotyping-by-sequencing. Extracted DNA mass ranged from 5.05 to 698.98 ng (mean = 90.01 ng), and all samples yielded viable libraries and produced high-quality data, with an average of 13.3 million reads per sample and 82.9% average alignment. Regression models showed that increased DNA mass significantly improved sequencing output in samples with DNA mass ≤ 150 ng. However, logistic regression indicated that only 13.6 ng of input DNA was required to achieve an 80% probability of sequencing success, with empirical success observed in all samples above ~ 40 ng. This is a substantial reduction in the amount of DNA usually requested in reduced representation sequencing protocols. These results provide practical guidance for low-yield genomic sampling and demonstrate that buccal swabs are compatible with reduced-representation sequencing protocols. Because these results reflect a specific combination of collection, extraction, and sequencing methods, success may vary under different workflows, but overall demonstrate the feasibility of low-input genomic sampling when optimized protocols are used. This approach offers a feasible alternative to invasive sampling for genomic studies of conservation-sensitive species, expanding opportunities for ethically grounded, field-based research.