design: Isolates were streaked from freezer stocks on 7H11 grown for 3-5 days at 37C then cultures grown in 7H9 supplemented with glycerol, OADC, and 0.05% tween 80. Morphotyping was done by visual examination on the primary streak plate. If multiple colony morphotypes were present, they were isolated and stocked. Isolates were grown from a single colony in 10mls. Cultures were centrifuged and DNA isolated using the MasterPure Gram Positive DNA Purification kit (Lucigen) with modifications*. Briefly, bacteria were resuspended in TE, transferred to Lysis Matrix B tubes, and 2 ul of lysozyme added and incubated for 1 hour at 37C. GP lysis solution was added at 300 ul per sample and tubed bead beat 3 time for 45 seconds each, placing the samples on ice to cool after each bead beating interval. Next 2 ul of proteinase K was added, heated to 65-70C for 15 minutes, the incubated at 55C for 2 hours. Samples were cooled to 37C, 350ul of MPC Protein and vortexed. Debris was pelleted by centrifugation at 4C for 10 minutes at >10,000 x g. To further remove debris supernatant was transfer to a new tube and recentrifuged before transferring to a fresh tube. Samples were depleted of RNA using 2 ul of RNaseA and incubating at 37C for 30 minutes. The resulting DNA was precipitated by adding 700 ul of isopropanol inverting to mix, centrifuging for 10 minutes 10,000rpm at 4C. Isopropanol was removed, and DNA pellet washed twice with 70% ethanol, dried, and resuspended in 100 ul of dH2O. DNA integrity was validating by running on an agarose gel and concentration quantified by qubit and sequenced by MIGS or SeqCoast where libraires were generated using the Illumina DNA Prep kit and custom IDT 10bp unique dual indices (UDI) with a target insert size of 320 bp. Sequencing was performed on an Illumina NovaSeq 6000 sequencer in one or more multiplexed shared-flow-cell runs, producing 2x151bp paired-end reads.