Genome sequencing of GP74
MinION sequencing libraries were prepared using the Ligation Sequencing Kit SQK-NBD-112.24 protocol version NBE_9134_v112_revL_01Dec2021 (last updated 15th March 2023) (ONT, Oxford, UK). 8 isolates were multiplexed per sequencing run. Starting with 400 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). 98 ng of each DNA repair and end-prepped sample was ligated separately with unique native barcodes and pooled for a final library of 508 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 2.3 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 v6.5.7 (Oxford Nanopore Technologies, 2021).