Purelink genome dna mini kit

Twenty-five ESBL-producing E. coli isolates were chosen for whole-genome sequencing (WGS). The isolates were confirmed to be ESBL-producers using the VITEK®2 compact system (bioMérieux, Nürtingen, Germany). DNA was isolated using the Purelink Genome DNA Mini kit (Invitrogen, Darmstadt, Germany). WGS was carried out on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) using an Illumina Nextera XT library with 2x300-bp paired-end reads. The data was assembled using SPAdes (version 3.0) (Bankevich et al., 2012 (link)). Contigs larger than 500-bp and a coverage higher than nine were ordered to E. coli MG1655 (accession number U00096.3) using MAUVE (Darling et al., 2004 (link)). The contigs were concatenated to generate pseudogenomes. Whole-genome phylogeny was determined by the software package Harvest Suite using E. coli MG1655 as reference (Treangen et al., 2014 (link)). The phylogenetic tree was drawn using MEGA5 (Hall, 2013 (link)).
The raw data of the sequenced E. coli are available at the European Nucleotide Archive (ENA), under the project number PRJEB12335.

Genomic DNA from SF100 WT and a DAP-resistant variant isolated on day 20 of passage (strain D20) from the VGS strain-set was isolated by cetyltrimethylammonium bromide (CTAB) extraction as described previously [15 (link),20 ]. Genomic DNA was further purified for sequencing using a PureLink Genome DNA Minikit (Invitrogen Waltham, MA, USA) according to the manufacturer’s instructions [15 (link),20 ]. PacBio library construction, sequencing, and annotation were conducted by using standard methods [15 (link),20 ]. The generation of genome assemblies and subsequent identification of single nucleotide polymorphisms (SNPs) and insertion–deletion mutations (indels) were performed as detailed elsewhere [15 (link),20 ]. All mutations identified by whole genome sequencing were confirmed by PCR and Sanger sequencing.

Eleven ESBL-producing isolates obtained from fish and 13 E. coli from the environmental samples were chosen for whole genome sequencing (WGS). DNA was isolated using the Purelink Genome DNA Mini kit (Invitrogen, Darmstadt, Germany) according to the manufacturer's instruction. WGS was carried out using an Illumina Nextera XT library with 2x300bp paired-end reads on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). The raw data was assembled using SPAdes (version 3.0) (Bankevich et al., 2012 (link)). Contigs from E. coli isolates were ordered by using MAUVE (Rissman et al., 2009 (link)) and E. coli MG1655 (accession number U00096.3) were chosen as a reference for all E. coli isolates, Citrobacter freundii strain P10159 for Citrobacter braakii isolates (accession number CP012554; no complete genome available for C. braakii), Klebsiella pneumoniae strain ATCC BAA-2146 (accession number CP006659) for K. pneumoniae and Enterobacter cloacae strain 34977 (accession number CP010376) for E. cloacae isolates. Pseudogenomes were created and whole genome phylogenetic analysis was subsequently performed by using ParSNP package of Harvest Suite (Treangen et al., 2014 (link)). The raw sequencing data of the sequenced isolates are available at the European Nucleotide Archive (ENA) under the project number PRJEB12361.