Bacterial DNA Extraction and 16S rRNA Amplification

We extracted bacterial DNA from swabs using DNeasy PowerSoil DNA isolation kits (Qiagen), consistent with previous work in this field^{22 (link)–24 (link)}. Extractions were carried out in 14 batches of 24 (23 uropygial gland swabs plus one swab-only negative control per batch, including swabs used as part of separate studies; details in^{22 (link),24 (link)}). We amplified the V4 region of the bacterial 16S rRNA gene using the universal primers F518^{77 (link)} and R806^{78 (link)}. Each primer included an Illumina MiSeq adaptor sequence, four randomized nucleotides, and a unique ‘barcode’ of eight nucleotides. We performed PCR in a total volume of 25 µL, including 10 µL of 5PRIME HotMasterMix (Quantabio), 0.2 µM of each primer, and 2 µL of DNA template ( $\overline{\text{x}}$ concentration = 0.1 ng/µL, range = 0.01–0.12 ng/µL, measured using a Qubit fluorometer). The thermocycling profile was: 2 min at 94 °C; 35 cycles of 45 s at 94 °C, 60 s at 50 °C, and 90 s at 72 °C; and a 10 min final extension at 72 °C.