16S (V4) amplicon sequencing of canine feces
Genomic DNA was extracted from frozen fecal samples using the Qiagen DNeasy PowerSoil Pro Kit. The 16S rRNA gene (V4 region) was amplified via PCR with Platinum Hot Start Master Mix (Invitrogen, Carlsbad, CA, USA) and barcoded 806R/515F primers with Illumina adapters. Amplicons were pooled, purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA), and quantified using the Qubit dsDNA HS assay. Libraries were sequenced on an Illumina MiSeq platform using standard protocols outlined for the single-end300bp v2 sequencing kit and raw reads were de-multiplexed using Illumina factory software.