Ion torrent s5 pgm platform

The whole genome sequence (WGS) of two randomly selected NDM-producing K. pneumoniae isolates (213A and 248D) was obtained and analyzed by next generation sequencing. Specifically, following DNA extraction, the concentration of double strand (ds) DNA was measured with Qubit using Qubit ds DNA HS assay kit (Q32851, Life Technologies) and NGS was performed using the Ion Torrent S5 PGM Platform (Life Technologies Corporation, Grand Island, NY, USA); shearing, purification, ligation, barcoding, size selection, library amplification and quantitation, emulsion PCR and enrichment were performed according to manufacturer's instructions and the products were loaded on a 316 chip. The Ion PGMHi-Q (200) chemistry (Ion PGM Hi-Q Sequencing kit, A25592) was applied (Thermo Fisher Scientific).
Identification of multi-locus sequence type (MLST), plasmids and antimicrobial resistance genes was performed applying the tools using MLST 2.0, Resfinder 4.1, KmerResistance 2.2, Comprehensive Antibiotic Resistance Database (CARD), and PlasmidFinder 2.1 [22] (link)[23] (link)[24] (link)[25] (link)[26] (link).

DNA was extracted using the DNA extraction kit (Qiagen, Hilden, Germany). The Qubit double strand (ds) DNA HS assay kit (Q32851, Life Technologies) was used for measurement of dsDNA concentration. WGS was performed in the Ion Torrent S5 PGM Platform (Life Technologies Corporation, Grand Island, NY, USA). All procedures regarding shearing, purification, ligation, barcoding, size selection, library amplification and quantitation, emulsion PCR and enrichment were conducted according to manufacturer's guidelines. PCR products were loaded on a 316 chip. The Ion PGMHi-Q (200) chemistry (Ion PGM Hi-Q Sequencing kit, A25592) was applied.