16S rRNA Gene Amplicon Sequencing

PCR amplicons for the V4 region of the 16S rRNA gene were generated with primers 515F–806R^{63 (link)} and were sequenced with the Illumina MiSeq 2 × 250 v2 Kit at the Cornell University Institute for Biotechnology. DADA2^{27 (link)} was used to call 100% sequence identity OTUs (i.e., sequence variants). Taxonomy was assigned to OTUs with the QIIME2 q2-feature-classifier^{64 (link)} using the SILVA database (v119)^{65 (link)}. The phyloseq^{66 (link)} R package was used to rarefy total OTU counts to 5000 per sample due to the multiple orders of magnitude difference in raw counts among samples. A phylogeny was inferred for all OTU sequences with fasttree^{67 (link)} based on a multiple sequence alignment generated by mafft^{68 (link)}. All samples lacking metadata used in the study were filtered from the dataset. In cases where an individual host was sampled multiple times, we randomly selected one sample.