Abstract The canine gut microbiome is an important model for microbiome research, yet methodological variation in DNA isolation, library preparation, and sequencing complicates cross-study comparisons. Here we present a three-component dataset to evaluate methodological effects. First, an ultra-deep sequencing dataset was generated from a single dog fecal sample using both short- (Illumina NovaSeq) and long-read (Oxford Nanopore MinION) platforms. Second, fecal samples from eight co-housed dogs