G tube fragmentation

DNA was extracted from bacterial isolates using the Agencourt GenFind DNA Isolation Kit and the Biomek FX^P Automated Workstation (Beckman Coulter). A short-read NGS library was prepared using the Nextera DNA Flex or XT Library Prep Kit (Illumina, San Diego, CA, USA). DNA quantification was performed in a microplate using the SpectraMax Quant dsDNA Assay Kit and the Gemini XPS Fluorometer (Molecular Devices, San Jose, CA, USA), or in a single tube using the Qubit 2.0 fluorometer (Thermo Fisher Scientific, Springfield Township, NJ, USA). Library quality was examined using the 4200 TapeStation and D1000 ScreenTape (Agilent, Santa Clara, CA, USA). Paired-end sequencing (2 × 150 cycles) was performed using the NextSeq 550 system (Illumina). The Pacific Biosciences (PacBio, Menlo Park, CA, USA) RSII single-molecule real-time (SMRT) sequencing system was employed for the long-read sequencing of A. baumannii PB364. The long-read NGS library was processed using g-TUBE fragmentation (Covaris, Woburn, MA, USA), BluePippin size selection (Sage Science, Beverly, MA, USA) and a SMRTbell template preparation kit (PacBio). Mainly, the manufacturers’ standard protocols were followed.

Genomic DNA was extracted from each isolate using a QIAamp DNA kit (Qiagen, Frederick, MD, USA) and subjected to both short- and long-read massively parallel sequencing. Short-read sequencing (150 bp × 2) was performed in the NextSeq system using the Nextera XT sample preparation kit (Illumina, San Diego, CA, USA). Genome coverage was reached at ~24× (PB367) and ~20× (PB350) on average. Long-read sequencing was performed using the PacBio RSII SMRT sequencing system. The SMRTbell library was processed using g-TUBE fragmentation (Covaris, Woburn, MA, USA), the BluePippin system for size selection of DNA fragments ranged from 7 to 50 kb (Sage Science, Beverly, MA, USA), and the SMRTbell template preparation kit (PacBio, Menlo Park, CA, USA). Genome coverage was reached at ~110× (PB367) and ~125× (PB350) in average.