Exp nbd104 kit

To determine the whole genome sequences, genomic DNA was extracted from 5 days’ bacterial culture of the strain TYQ2. The DNA library was constructed by Nanopore PromethION platform and Illumina NovaSeq platform at the Beijing Novogene Bioinformatics Technology Co., Ltd. Firstly, large DNA fragments were recovered by Blue Pippin automatic nucleic acid fragment recovery system, and then repaired. Next, barcode was added by PCR-free method of EXP-NBD104 kit from Oxford Nanopore Technologies Company. The fragments’ size was detected by AATI automatic capillary electrophoresis instrument to get the samples isomolarly mixed. Afterward, the SQK-LSK109 connection kit was used to connect the adapter and the library was preliminarily constructed. Then, sequencing libraries were generated by using NEBNext^® Ultra™ DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, United States). PE150 data and Nanopore data were combined to assemble by using Unicycler.

MinION (Oxford Nanopore Technologies) was chosen as the LR-NGS platform and WGS was performed on 20 isolates (DNA samples of three isolates were not available). For library preparation, DNA was end-repairelid and A-tailed with NEBNext FFPE DNA Repair Mix and the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Ligation of the sequencing adapters was performed using the SQK-LSK109 kit (Oxford Nanopore Technologies) and each sample was barcoded using the EXP-NBD104 kit (Oxford Nanopore Technologies). Each step was followed by purification with Agencourt AMPure XP beads (Beckman Coulter). Libraries were pooled in equimolar amounts and adjusted to a final concentration of 1 pM. Twenty isolates were sequenced per flow cell (FLO-MIN106D; Oxford Nanopore Technologies) using MinKNOW software (ver. 20.10; Oxford Nanopore Technologies).