Applicability of Non-Invasively Collected Eurasian Goshawk (Astur gentilis) Moulted Feathers for Whole Genome Sequencing Analysis

<b>Background/Objectives</b>: Non-invasive samples offer an attractive alternative to logistically challenging invasive approaches in wildlife genetic studies but often contain low-quality host DNA that limits downstream analyses. Here, we assessed the applicability of moulted Eurasian goshawk feathers as a DNA source for whole-genome re-sequencing. <b>Methods</b>: We analysed 75 moulted feathers collected opportunistically from breeding territories. Each feather was measured from tip to tip, and its condition was visually assessed. Whole-genome re-sequencing was performed with a target coverage of 13× using 150 bp paired-end reads. <b>Results</b>: Feathers yielded an average of 7.19 ± 10.93 ng/μL DNA. DNA yield was positively correlated with feather size and the presence of blood traces in the calamus. On average, feather samples performed well, producing 208.7 ± 59.82 million reads, of which 82.69 ± 27.15% aligned to the reference genome, resulting in 83.58 ± 19.02% of the genome being covered at least once. After quality filtering, 10.34 ± 3.11 million biallelic single-nucleotide variants remained, of which 457,745 were common variants (MAF > 0.05). Larger feathers in good condition, with higher DNA yields and blood traces in the calamus, tended to perform better throughout the re-sequencing workflow. Nevertheless, approximately 22.7% of samples failed due to high missing data or poor genotype quality. <b>Conclusions</b>: Performance varied substantially even among samples with similar characteristics, indicating that improved sample selection incorporating direct measures of host DNA quality may be beneficial. Despite these challenges, moulted feathers represent a readily available DNA source for genome-wide re-sequencing of medium- to large-sized raptor species.