Microbiology diagnostic laboratory plays a significant role in public health surveillance, outbreak investigation, infection prevention and control strategies. It is moving towards incorporating molecular biology techniques for the surveillance and identification of pathogens causing infectious diseases. Next-generation sequencing (NGS) holds potential for improving clinical and public health microbiology. In addition to identifying pathogens more rapidly and precisely than traditional methods, sequencing technologies can provide new insights into disease transmission, virulence and antimicrobial resistance. NGS has not only reduced the cost of total sequencing but has also introduced versatile applications under one platform. This review will discuss the methods, principles and applications of genome sequencing in microbiology diagnostic laboratories.

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The precise diagnosis of fungi is utmost important owing to the morbidity and mortality caused especially in various susceptible hosts. Among the diagnostic methods though the phenotypic methods are being routinely used among laboratories but they have inherent hindrances of being tedious, time-consuming and entail experience. These roadblocks acting as a major obstacle in precise identification of fungi, underlined the requisite for implementation of genotypic methods for routine diagnosis. Since sequencing forms the cornerstone of molecular identification of fungi, many sophisticated modalities and platforms have been developed. The role of fungal sequencing isn't limited merely to the identification of known fungal species in routine laboratory, but is of utmost significance in deciphering the emerging pathogenic and saprophytic fungal species that have the potential to infect humans. It was with the use of these sequencing techniques that the complex fungal nomenclature based on presence or absence of sexual form of the fungus, could be simplified and unified effort led to adoption of 'one-fungus--one-name' rule. Panfungal PCR targeting 28S rRNA when used in conjunction with sequencing for detection of etiological agents in patients with invasive fungal disease (IFD) from deep tissue samples has shown encouraging results. Though many sequencing modalities are available, an ideal diagnostic platform is yet awaited to meet the diversity of fungal infections in initial stages itself. The early diagnosis enables the clinician to administer appropriate therapy as and when required. The same helps in delimiting the undesired affects of antifungals as well as indirectly help in antimicrobial stewardship as well.

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Gene sequencing is the inevitable future of diagnostic microbiology. Of the various molecular assays available, sequencing is the promising technique for detecting culture-negative infections due to uncultivable bacteria namely culture-negative endocarditis, meningitis, brain abscess, keratitis, urinary tract infections, empyema, septic arthritis and septicaemia. Sequencing also helps to predict full resistance profile of bacteria and its virulence traits. Sequencing is an emerging and powerful technique to perform the epidemiological studies in an outbreak situation. This review focuses on the common applications of sequencing in clinical bacteriology including isolate characterisation, antimicrobial resistance and virulence factor profiling, establishing the source of infections and tracking the disease transmission.

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Parasitic infections and its burden are increasing worldwide and there are many unknown areas among the parasites for a long time which makes the eradication of most of the parasites impossible till now. The mechanism of how certain parasites evade human immune response and how they escape from the action of antiparasitic drugs were unclear. It is also difficult to maintain their culture in the laboratory which makes it difficult to identify to the species level most of the time. The field of sequencing has undergone many advances recently. The availability of entire genome sequences has revolutionised the study of infectious organisms, including parasites. It helps us to know the complete nucleotide sequence of even a complex genome at larger number. In the form of pilot genome sequencing studies, genomics approaches have been used to deal with the problem of tropical neglected parasitic illnesses for more than 20 years. In this, new technology like sequencing is coming up in a big way not only in the diagnosis but also targeted therapeutics and its control. Hence, different sequencing methods have been explored briefly in its role in parasitic diseases.

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An objective method of detecting infections without the requirement for clinical hypotheses is provided using next-generation sequencing (NGS) technology for the diagnosis of infectious disorders. In order to inform nations and the general public about any potential changes that may be required to respond to the variant and stop its spread, the World Health Organization and its international networks of experts have been continuously monitoring changes to the virus based on NGS data throughout the current pandemic. When tracking ongoing outbreaks, monitoring for novel pathogens or spotting potentially harmful variations of well-known diseases, NGS offers substantial advantages. Due to the technology's quick creation of high-resolution sequence data, researchers and research teams can access it easily and share it with one another. Numerous candidate vaccines have been created on several platforms quickly. Many of them have worked in crisis circumstances in numerous nations worldwide. The breakthrough infections could only be tracked by the use of NGS technology. In this review, we discussed in silico analysis using current bioinformatics approaches, and sequencing reveals unique emerging severe acute respiratory syndrome coronavirus 2 variations which have the potential to cause novel illnesses in the future.

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Next-generation sequencing (NGS) is a robust platform which can be employed for pathogen detection. It has the potential to identify pathogens in an unbiased nature including bacteria, fungus, parasites, DNA/RNA viruses and even newer novel pathogens which have been previously reported. The high sensitivity of this technology makes it suitable for ophthalmology applications that rely on very small clinical specimen volumes. Currently, the diagnosis of ocular pathogens relies mainly on conventional procedures such as culturing and microscopy. Traditional methods lack sensitivity, which can be compensated by the application of molecular tools, such as polymerase chain reaction and NGS. In this review, we will look at how NGS can be used to treat ocular infectious diseases such as keratitis, conjunctivitis, trachoma, dacryocystitis, lacrimal sac infection, endophthalmitis and uveitis, with added information on the limitations, advantages and disadvantages on the ocular implication of this NGS technology. NGS-based approaches increase the sensitivity of pathogen detection as well as have the potential to improve healthcare services. NGS still requires advancement in the data analysis tools, establishing standards and reducing turnaround time. We are now closer to realising the full potential of this high-throughput technology. Importantly, NGS also delivers additional data which gives a unique opportunity to explore potential biomarkers of disease pathogenicity or underlying genetic predisposition.

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