Whole-Genome Sequencing of Acinetobacter Strains

Genomic DNA was extracted using the Wizard Genomic DNA Extraction Kit (Promega, Fitchburg, WI). The quality and concentration of the DNA were assessed using a Nanodrop bioanalyser spectrophotometer (Thermo Scientific, Delaware, USA). All isolates were sequenced on an Illumina MiSeq using the Nextera XT as per the manufacturers’ protocols. All samples were multiplexed and sequenced on a single MiSeq 2 × 151 bp run (EBI: PRJEB10709). Demultiplexed FASTQ files were quality controlled using Trimmomatic-0.30 [LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36]40 (link). Draft genomes were de novo assembled using VelvetOptimiser-2.2.5 (Victorian Bioinformatics Consortium, Australia) and Velvet 1.2.0941 (link). Contigs from the sensitive parental strains were ordered by Abacas 1.3.142 (link) using A. baumannii MDR-TJ (CP003500) as a reference. Contigs breaks were joined using IMAGE43 (link). Derived progeny draft assembly contigs were ordered to the parental strain using Abacas. Genomes were annotated using Prokka and an custom Acinetobacter reference library44 (link). Laboratory induced SNPs were identified by mapping progeny sequencing reads against the parental isolate using BWA45 (link) and SAMtools46 (link) [varFilter.pl]. All SNPs were manually checked. MLST was determined using BLAST47 (link) and a custom allele database.