title: CFIA-AMR-POOL-0004
design: Frozen stonefruit samples (25 g) were enriched in BPW and incubated for 18-24 h at 37C. Enrichments were then diluted and plated on selective differential agar containing antibiotics, and incubated overnight at 37C. Colonies of different morphologies from selective agar were then purified and enriched individually in brucella broth for 18-24 h at 37C. Equal volumes (100 L) of up to 25 purified isolate enrichments were then pooled together, centrifuged at 8,600 RPM, and resuspended in 300 L of fresh brucella broth. Only 25 isolates were pooled as this method aimed to achieve at least 5-10X coverage of each isolate. Genomic DNA was extracted from 300 L of the resuspended pool using the Maxwell RSC Cultured Cell DNA Purification kit (Promega, Madison, WI). DNA was quantified using the Quant-iT High-Sensitivity DNA Assay Kit (Life Technologies Inc., Burlington, ON).  Pool-sequencing (pool-seqs) was performed on the Illumina MiSeq Platform (Illumina, Inc.) using a 600-cycle MiSeq Reagent kit v3 (Illumina, Inc.). Sequencing libraries were constructed from 1 ng of gDNA using Nextera XT DNA Sample Preparation and Index Kits (Illumina, Inc., San Diego, CA) according to manufacturers instructions. Data from pool-seqs was used as a high-throughput screening method to investigate antimicrobial resistance genes of interest and taxonomy in the bacterial isolates from stonefruit samples.