Abstract
Next-Generation Sequencing (NGS) is becoming a reality in the clinical microbiology laboratory because it can speed diagnosis when compared to traditional culture based-methods and moreover, to aid in unravelling key virulence traits of important pathogens. Nonetheless, there are many limitations for its wide application in routine testing, as the requirement of high performance hardware and software to support bioinformatics analysis, as well as the expertise in different programming languages to perform the analyses. In this context, this review was drawn to synthesize some basic concepts involved in NGS for Whole-Genome Sequencing (WGS), based on two international straightforward efforts to standardize WGS data acquisition and processing in the clinical routine, the PulseNet International and the ENGAGE project, allied with other tools available for WGS analysis, beginning from the available sequencing platforms to the main user-friendly pipelines dedicated for the pathogen identification, including the use of properly databases to search for virulence factors, resistance genes and software resources for molecular typing of isolates.
Highlights
The diagnosis of bacterial pathogens of clinical importance is still very dependent on phenotypic techniques, which include plate culture for isolation of colonies, differential staining, morphological analysis and biochemical tests [1]
The aim of this manuscript is to address the application of Whole-Genome Sequencing (WGS) for molecular identification of bacterial isolates, we must point out that through the Next-Generation Sequencing (NGS) technology it is possible to perform analyses of clinical samples by independent culture methods such as metagenomics and metataxonomics, where the first is based on the sequencing of the genomes of all the microorganisms present in the sample, returning a set of annotated theoretical genomes, while the second one refers to the sequencing of specific regions of the 16S rRNA gene present in the microbial genomes of a given sample [58]
Even with so many different applications of the WGS for diagnostic microbiology, barriers related to the interpretation of data by microbiologists, which rely on bioinformaticians for data analysis, creates a major limitation for NGS technology to be incorporated into routine laboratories
Summary
The diagnosis of bacterial pathogens of clinical importance is still very dependent on phenotypic techniques, which include plate culture for isolation of colonies, differential staining, morphological analysis and biochemical tests [1]. The ENGAGE (Establishing Generation sequencing Ability for Genomic Analysis in Europe – www.engageeurope.eu) project, composed by eight European institutions, published SOPs for genomic data collection from a pure culture isolate, WGS DNA sequencing and data analysis suggesting dedicated tools for quality control, genome assembly and annotation viewing to standardize pathogen identification in different laboratories across Europe.
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