E gel sizeselect 2 agarose kit

A subset of the clinical isolates was analyzed using whole-genome sequencing (WGS). The bacterial genomes were sequenced using the IonTorrent PGM platform (Life Technologies, Carlsbad, USA) in accordance with the manufacturer’s instructions. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies), and its size was selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analyzed using the High Sensitivity kit (Agilent, Santa Clara, USA). Further sample processing was performed using the Ion OneTouch kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). The single reads obtained were de novo assembled using MIRA 3.9.9, which is a part of the Assembler plugin on the Ion Torrent server. The contigs were analyzed using the ResFinder version 4.1. web-service [80 (link)].

Isolates were transported to the Molecular Diagnostics group at the Austrian Institute of Technology where they were checked for viability at arrival. Fifteen isolates were not viable after transportation, and sequencing failed in three isolates. After a viability check of all isolates, genomic DNA was extracted with the QiAmp DNA mini kit (Qiagen, Hilden, Germany). Whole genome sequencing (WGS) was performed using the Ion Torrent PGM platform using 400 bp read chemistry. Sequencing was performed according to the protocol recommended by Life Technologies. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies, Carlsbad, CA, USA) and its size selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analysed using the High Sensitivity Kit (Agilent, Santa Clara, Santa Clara, CA, USA). Further sample processing was performed using the Ion OneTouch Kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). Raw reads were assembled de novo using Assembler SPAdes software [7 (link)]. The genome was annotated using the RAST (Rapid Annotations using Subsystems Technology) database [8 (link),9 (link)].