Blood and tissue genomic dna extraction kit

Genomic DNA (gDNA) was extracted from Listeria isolates using the blood and tissue genomic DNA extraction kit (Qiagen, Germantown, MD, United States); purity of extracted gDNA was assessed using a NanoDrop™ spectrophotometer. The concentration of gDNA was determined using a Qubit^® double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Life Technologies Inc., Carlsbad, CA, United States) on an Invitrogen Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) according to manufacturer’s instructions. Sequencing libraries were prepared using Nextera™ XT DNA Sample Preparation Kit and Nextera™ XT Index Kit (Illumina Inc., San Diego, CA, United States). Libraries were sequenced on an Illumina MiSeq platform using a MiSeq v2 reagent kit (Illumina Inc., San Diego, CA, United States) with 500 cycles to generate a paired-end read length of 2 × 250 bp. Quality check of raw reads was determined using FastQC tool,¹ and low quality reads were trimmed using trimmomatic with the following parameters: leading: 10, trailing: 10, sliding window: 4:20, and minlen: 40. Obtained reads were then de novo assembled using A5-miseq assembler (Coil et al., 2015 (link)) and assembly statistics that included genome length, number of contigs, coverage, GC% and N50 were determined.

Isolates used in this analysis (n = 18) were selected from our previous study and included E. cecorum from non-clinical poultry collected by the animal arm of the National Antimicrobial Resistance Monitoring System (NARMS) from 2003–2011 and from diseased chickens from clinical cases submitted between 2008–2011 to the Animal Diagnostic Laboratory at the Pennsylvania State University [21 (link)]. E. cecorum genomic DNA was extracted using the blood and tissue genomic DNA extraction kit (Qiagen, Germantown, MD, USA). Extracted genomic DNA (gDNA) was quantified using Qubit double-stranded DNA (dsDNA) high-CHS) assay kit according to the manufacturer’s instructions (Life Technologies Inc., Carlsbad, CA, USA). The quality check of gDNA was performed using a NanoDrop™ spectrophotometer.