Genome sequencing of GP76
MinION sequencing libraries were prepared using the Ligation Sequencing Kit SQK-NBD-114.24 protocol version NBE_9169_v114_revJ_15Sep2022 (last updated 7th September 2023) (ONT, Oxford, UK). 11 isolates were multiplexed per sequencing run. Starting with 1000 ng 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). 288 ng of each DNA repair and end-prepped sample was ligated separately with unique native barcodes and pooled for a final library of 1806 ng, and adapters were ligated to the pooled 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 10-20 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 fast5 data converted from raw pod5 files with the ONT software Dorado v0.3.4 (Oxford Nanopore Technologies, 2021). Diluted DNA control sample added to each DNA sample was filtered out with the BBDuk function in the BBTools package version 39 (https://bbmap.org)