Genome sequencing of GAS15
MinION sequencing libraries were prepared using the Ligation Sequencing Kit SQK-LSK-109 (ONT, Oxford, UK). Between 6-8 isolates were multiplexed per sequencing run using the Native Barcoding Expansion Kit EXP-NBD104 (ONT, Oxford, UK). We skipped the Covaris g-TUBE shearing step to target long fragment DNA. Starting with 1 g of DNA per isolate, samples were prepped with the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-Tailing kits (New England Biolabs, Ipswich, MA, USA), and DNA cleanup was performed using Ampure XP beads (Beckman Coulter, Pasadena, CA, USA). 150 ng of each barcoded sample was pooled for a final library of 700-1000 ng, and adapters were ligated to the sample with Blunt/TA ligase (New England Biolabs, Ipswich, MA, USA). The long fragment buffer was used in an extended 10 minute incubation at 37 C to enrich for high molecular weight DNA. The flow cell was primed using the Flow Cell Priming Kit (ONT, Oxford, UK). Approximately 15 fmols of the pooled library, sequencing buffer, and loading beads were mixed and then loaded through Spot-On port of the flow cell. Sequencing runs completed after 72 hours, and high accuracy base calling was performed on the raw fast5 data with the ONT software Guppy v4.5.4 or v6.1.1 (Oxford Nanopore Technologies, 2021).