Abstract
Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.
Highlights
Identification and characterization of micro-organisms that cause infections are crucial for successful treatment, recovery and safety of patients
Pacific Biosciences use fluorescent nucleotides in their single molecule real-time (SMRT) technology, and Oxford Nanopore platforms use ionic current sensing, in which DNA is guided through nano-pores, thereby changing the current in a way that is specific for the type of nucleotide
Next generation sequencing (NGS) is already applied in several medical microbiology laboratories, including our laboratory at the University Medical Center Groningen (UMCG), where it is used for outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches
Summary
Identification and characterization of micro-organisms that cause infections are crucial for successful treatment, recovery and safety of patients. Important role in medical microbiology laboratories (Buchan and Ledeboer, 2014) These methods have reduced the turnaround time from receiving the sample to the final result, and made it possible to detect non-cultivable pathogens. One of the molecular methods used in medical microbiology laboratories is the sequence analyses of genes or the whole genome of pathogens. The same method can be applied for the identification of pathogens in clinical material This approach becomes problematic when clinical material is more complex and contains multiple species, such as faecal samples. In such cases, results obtained by Sanger sequencing are not reliable and make it hard or even impossible to identify specific pathogens. NGS is requested by clinical microbiologists or infectious disease specialists in collaboration with molecular microbiologists and infection control practioners
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