Mock sample comprised of homogenate from hoof lesions
Skin biopsy punches were preserved in 1 mL Qiagen All-Protect (Qiagen, Germantown, MD, USA) and frozen at -80C until processed.   At processing, samples were thawed on ice, removed from cryovials, and blotted to remove excess liquid.  DNA was extracted using Qiagen DNeasy Blood and Tissue kit following manufacturer instructions for tissue with overnight digestion.  Total DNA yield was quantified with a Qubit fluorometer (Life Technologies, Grand Island, NY) and DNA stored at -80C.  Amplicon libraries were prepared for bacterial 16S rRNA gene v4 region sequencing as described (https://www.anl.gov/bio/environmental-sample-preparation-and-sequencing-facility).  Sequencing was performed using Illumina MiSeq v2 chemistry and universal 16S rRNA gene forward primers (515 F) and unique Golay barcoded reverse primers (806R).