Minion

Whole-cell genomic DNA of ATCC 19606 was isolated using the DNeasy^TM UltraClean Microbial Kit (Qiagen^TM, Germantown, MD, USA) from cells grown overnight at 37 °C in LB inoculated from a single colony. Library preparation and barcoding for Illumina MiSeq and MinION (Oxford Nanopore Technologies ^®, Oxford, UK) sequencing was performed by the UTS Core Sequencing Facility at the ithree Institute, as described previously [15 (link),16 (link)]. Illumina sequencing generated 1,024,087 paired-end short reads with 50-fold coverage and an average length of 250 bp; MinION generated a total of 10,687 reads with an N50 of 18.2 kbp and 30-fold coverage. FastQC (v.0.11.9) (https://bioinformatics.babraham.ac.uk/projects/fastqc/) and Filtlong (v.0.2.0) (https://github.com/rrwick/Filtlong) were used to check the quality of Illumina and MinION reads, respectively. Filtlong filtered long reads by quality and length. The high=quality Illumina and MinION reads were assembled de novo using a hybrid assembly approach with the Unicycler program (v0.4.7) [17 (link)]. Protein coding, rRNA and tRNA gene sequences were annotated using Prokka [18 (link)], and the resistance and polysaccharide loci (outlined below) were annotated manually.

Genomic DNA was extracted from overnight yeast cultures by zymolyase digestion and standard phenol-chloroform extraction (Treu et al., 2014 (link)). A combined sequencing approach was then applied using Illumina and Oxford Nanopore MinION single molecule sequencers. Illumina library was generated using the TruSeq DNA PCR-Free Library Prep Kit (Illumina Inc., San Diego CA) and Covaris S2 (Woburn, MA) for a 550-bp average fragment size. Library was loaded onto the flow cell provided in the NextSeq 500 Reagent kit v2 (150 cycles) (Illumina Inc., San Diego CA) and sequenced on a NextSeq 500 (Illumina Inc., San Diego CA) platform with a paired-end protocol and read lengths of 151 bp at the CRIBI Biotechnology Center (Padova, Italy). Nanopore library was prepared according to SQK-LSK109 ligation sequencing kit and sequenced on a FLO-MIN106 R9 flowcell.

Bacterial genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, United States), followed by the MiSeq (Illumina, United States) and MinION (Oxford Nanopore, United Kingdom) sequencing. The short Illumina reads were trimmed to remove the poor quality sequences, and the resultant contigs were assembled using Newbler3.0 (Nederbragt, 2014 (link)). The longest single read obtained by the MinION sequencer was 98 kb, thereby crossing the repetitive shufflon regions in the plasmid (Laver et al., 2015 (link)). The long reads from the MinION combined with the short Illumina reads were hybrid assembled using SPAdesv3.11.1 (Bankevich et al., 2012 (link)). Hybrid assembly produced several scaffolds and BLASTN analysis confirmed that the scaffold in our study has the highest similarity to the plasmid p16Pre36-NDM (Accession no. KX832927) with coverage of 69% and identity of 96%. As most of the published plasmids are in a circle form, further bioinformatics analysis confirmed that this scaffold can be successfully cyclized using our in-house script. The correctness was then proved by mapping the high-throughput sequencing reads to the cyclized scaffold using CLC Genomics Workbench 9.0, with a mean reads mapping coverage of 111x. The consensus sequence acquired from CLC Genomics Workbench 9.0 was finally treated as the complete sequence of our plasmid pHFK418-NDM.