Sepsis is a major public health concern that is defined as a life-threatening organ dysfunction caused by a dysregulated host response to an infection. The most challenging issue in sepsis is early and appropriate diagnosis and treatment of it. Most of the routine clinical diagnosis methods are culture-based that take few days to get results. So, a method such as Next-generation Sequencing (NGS) which can generate large amounts of sequence data rapidly in a single run seems to be beneficial for sepsis diagnosis. Thus, this project aimed to analyse NGS data of bacterial isolates collected from suspected sepsis patients and previously identified as E. coli by the MALDI-TOF MS. The aim of NGS analysis was to identify species, virulence genes, and antibiotic resistance genes as well as comparing the genotypic antibiotic susceptibility results with phenotypic antibiotic susceptibility testing (AST). Also, the results of the in-house pipeline and a commercially available tool (1928 Diagnostics) were compared. Analysis of NGS data started with quality control and trimming of FASTQ files. Following, assembly and quality control of assembled files were performed. All isolates were analysed with the in-house pipeline as well as the 1928 Diagnostics platform to identify species, virulence genes, antibiotic resistance genes, and phylogenetic. Analysis of Multi Locus Sequence Typing (MLST) indicated that the most prevalent sequence types were ST73, ST95, and ST131. Analysis of virulence genes showed that most identified virulence genes belonged to the uropathogenic strain. The antibiotic resistance analysis illustrated that resistance to beta-lactam was prevalent among isolates. Moreover, an association between antibiotic resistance and sequence types was found. Also, comparing phenotypic AST and genotypic antibiotic susceptibility results indicated that discordance was observed between the results of the two methods. In conclusion, NGS analysis provided valuable information about extra-intestinal E. coli comparing routine clinical tests such as MALDI-TOF MS. The sequence type of the bacteria is one of the most important information that can be obtained by NGS analysis and is associated with resistance profiles of isolates. Regardless of discordance between two genotypic and phenotypic methods, the pipelines accurately predicted antibiotic resistance profiles of isolates in 92% of cases therefore they are efficient in NGS analysis. It should be noted that genotypic analysis of antibiotic resistance of isolates may give false-positive results while compared to phenotypic AST that should be considered in every NGS analysis.