Nebnext ultra 2 fs dna library preparation kit

Genomic DNA was purified from overnight bacterial cultures using Genomic-tip 100/G (Qiagen). Libraries for short-read sequencing were prepared with the NEBNext Ultra II FS DNA library preparation kit (New England Biolabs [NEB]) and sequenced using Illumina MiSeq apparatus to obtain 301-bp paired-end reads. Libraries for long-read sequencing were prepared with the Rapid Barcode kit (SQK-RBK004), sequenced using an R9.4.1 flow cell on the Oxford Nanopore MinION system, and base-called using Guppy GPU v.3.4.5 (ONT Nanopore). Hybrid assembly of short and long reads was performed using Unicycler v.0.4.8 to obtain complete genome sequences (29 (link)). Annotation was performed using dfast v.1.2.6 with default parameters (30 (link)), and the annotation of mgrB was manually curated using the reference sequence with accession no. ACI07312.1. SNPs, insertions, and deletions were identified by aligning each chromosome to that of KpWEA1 using MUMmer4 (31 (link)). Plasmid replicons were identified using PlasmidFinder 2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) with default parameters. Antimicrobial-resistance genes were searched using ResFinder 3.2 (https://cge.cbs.dtu.dk/services/ResFinder/) with default parameters. Prophages were identified using PHASTER (32 (link)). The complete genome sequences are available in DDBJ/EMBL/GenBank (accession numbers in Table 2).

Illumina sequencing libraries were prepared using NEBNEXT ULTRA II FS DNA library preparation kit (New England Biolabs), then multiplexed paired-end/single-end sequencing was performed using the HiSeq2500 NGS platform (Illumina, San Diego, CA, United States). Information on raw sequence data (registered under BioProject ID: PRJNA616081) is provided in Supplementary Table 1. The sequence reads were quality controlled and analyzed using previously described procedures (Brites et al., 2018 (link)).