Wizard genomic dna extraction kit

A total of ten isolates (three from outbreak one, five from outbreak two and two from environmental samples related to outbreak two) were subjected to DNA extraction using a Wizard genomic DNA extraction kit (Promega) [44 ]. A Qubit fluorometer was used for measuring DNA concentrations following DNA extraction. For WGS, 1 ng extracted DNA from each strain was used to create a sequencing library using the Nextera XT library preparation kit, as per the manufacturer’s instructions. WGS was performed on an Illumina MiSeq platform to generate 250 bp paired-end reads [45 ]. Raw reads were deposited in the European Nucleotide Archive (ENA) under study accession number PRJEB38898 (Table S1).

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.

The study was conducted in 18 different provinces representing five different geographical regions of Turkey (Fig. 1). A total of 1979 blood samples were collected from sheep (n = 1727) and goats (n = 252) between 2011 and 2013 from the selected provinces. Blood samples were collected in EDTA tubes from randomly selected animals in each herd that were at least one year of age. DNA was extracted from blood samples using the Promega Wizard genomic DNA extraction kit (Madison, WI, USA) following the manufacturer’s instructions. Extracted DNA was resuspended in 100 μl elution buffer and stored at -20 °C until analysed. Control DNA samples used in this study included isolates of T. ovis (Kayseri), Theileria sp. MK (Kayseri) and B. ovis (Kayseri) from Turkey, isolates of T. lestoquardi (Lahr) and B. crassa from Iran, isolates of T. uilenbergi (Longde), T. luwenshuni (Lintan) and B. motasi (Lintan) from China and an isolate of A. phagocytophilum from the United Kingdom.Fig. 1

Map of Turkey showing the provinces of five geographical regions where the samples were collected

Blood samples were collected at the Fourth Hospital of Hebei University from 180 ESCC patients who underwent tumor resection in the Department of Surgery between 2004 and 2008. Blood was also collected from age-matched healthy controls. Genomic DNA was extracted immediately with a Wizard Genomic DNA extraction kit (Promega, Madison, WI). All procedures were supervised and approved by the hospital’s Human Tissue Research Committee. Inform consent were provide to all patients enrolled in this study and the methods were carried out in accordance with the approved guidelines.

Genomic DNA from the NTS isolates was extracted using the Wizard Genomic DNA Extraction Kit (Promega, Fitchburg, Wisconsin). Two micrograms of genomic DNA was subjected to indexed whole genome sequencing (WGS) on an Illumina Hiseq 2000 platform at the Wellcome Trust Sanger Institute, to generate 100-bp paired-end reads. WGS was performed using new sequencing technologies (Illumina, 454) and exploited genotyping platforms (GoldenGate) for high-resolution and high-throughput sample analysis. The likelihood test ratios were determined as previously described [25 (link)]. The support for nodes on the trees was checked using 100 random bootstrap replicates. Resulting phylogenetic trees were visualized using the FigTree package version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). Subtrees were extracted for each subclade, which are therefore each rooted by the other subclades.