Next Generation Sequencing (NGS) in Clinical Microbiology

Next Generation Sequencing (NGS)

NGS is DNA sequence technology. It is also regarded as non-Sanger based high through put technology. Sanger sequencing for DNA is regarded as standard in DNA sequencing. Genome which was 1^st sequenced in 1977 was bacteriophage using Sanger technique.
All such recent modifications instrumentation that has increased the rate of sequencing called as high throughput sequencing. There may use of computational biology. It is processing ability of instruments. Early advancements made in early 1990s and late 1990s. The Capillary system in which the time for electrophoresis become short. There may be 96-300 capillary system instrumental processing has been used. Thus handle number of samples at a time.

All new recent techniques which were developed during last decade or recent are 1000 times more advance than Sanger method. By recent techniques one can sequence more than 2 Million base pair per day. These new recent techniques are termed as Next Generation Sequencing (NGS).
NGS technology has number of application in research and clinical diagnosis. Here we’ll discuss the applications of NGS in Microbiology.

Applications of NGS in Clinical Microbiology

It is obvious that NGS can replace the current clinical microbiology diagnostic laboratory techniques which are time and labor consuming. Some species of Mycobacterium and Bacteria, viruses are specialized even organism specific. So in traditional clinical laboratories consumption of immense resources still unable to produce desired and accurate results. In the end Microbiologist has to make guess about pathogenic activities, for diagnostic and microorganism identification. Thus results in the wastage of time both Doctor and patient. Thus here comes the need of NGS technology in clinical microbiology for diagnostics.

This technology can provide clinically actionable results sooner than traditional diagnostic labs.

Moreover NGS can provide a bold, innovative and conclusive decision about the treatment of a disease. Thus beneficial for both the patients and Drs.

In Clinical Microbiology Labs NGS is applied to

Outbreak Management

Despite concrete efforts, outbreaks still have a chance to prevail in a hospital or clinic. NGS helps in outbreak management, by timely sequencing the outbreak isolates, and provide proper guidance to infection control. Fast identification of infection causing pathogens and infectious containing sources is much important to outbreak management, and this could only be done via NGS. Because NGS provides fast pathogen detection, this results in quicker anticipation, like patient isolation, contact preventions and decolonization. NGS also provide WGS based typing which helps in epidemiological studies and health investigations. WGS is useful in identification of multi-drug resistance bacteria. Apart from this it can also helps in characterization of virulent microorganisms.

NGS can also helps in spread of infection within or in between the hospitals.

Molecular Case Finding

In Outbreak investigation usually we use the information obtained from molecular case outbreak isolates. NGS databases can be searched in comprehensive and more complex outbreaks for case findings. By doing so we may detect a case, which might not be possible with the traditional investigations of epidemiology. Studies have shown that, an enzyme was isolated from patient providing him a long facility under high care, and the patient has no recent history of abroad travelling, the name of the molecule is Metallo-ß-lactamase-5 (NDM-5)-producing K. So in this patient case finding on molecular bases have shown that the strain of patient was related clonally to other 4 patients strains isolated in 2014, these 4 patients were living in other country which was far away from the patient isolated the molecular case. In the hospitals of these countries no epidemiological links were found. So then contacts been made to molecular case finding via NGS databases they already developed, with European national surveillance centers, surprisingly there were no other cases were reported. Later than the finding of Plasmid Resistance Gene MCR-1 some other molecular case finding examples were reported to NGS database. In life-stock and hospitalized patients This Gene is responsible for colistin resistance. This is how NGS data base search was introduced in Europe in molecular case finding. After the revelation of the MCR-1 gene Enterobacteria case was isolated and screened. Thus new antibiotic resistance genes can be searched by using NGS techniques via molecular case findings.

Characterization of Pathogens

Currently the characterization techniques are based on bacteriological, biochemical and molecular methods which no doubt makes it laborious and time consuming. While NGS can be proved the best and fast one step characterization of pathogens their studies and applications. In beginning of a disease the crucial thong is to identify the profile of a virulent pathogen, in this way we can predict the severity, infection outcome, and risk assessment of the disease. By using NGS we can do WGS, and WGS is used for molecular characterization. Required information about the pathogen is obtained Insilco from the from the data sequence, like genotype, serotype, MLST profile, Virulence and antibiotic resistance profile, and even phylogenetic history, and overall molecular profile of a pathogen, and distinguish between strains which are closely related.

NGS helps us to characterize and distinguish between closely related strains within a very short duration of time. Thus to characterize and surveillance of pathogens the role of NGS and WGS cannot be denied.

NGS also allows in identification of novel Resistance genes, further their studies whether these genes are responsible for particular infection or not.

Targeted NGS of 16S-23S rRNA regions

Culture-free detection of number of Bacterial Pathogenic species is possible with NGS, so provides full micro biome insight. In clinical samples we want to detect the Bacteria, viruses and Fungi, so metagenomics would be the ultimate goal. So for this purpose computational biology skills are required which is usually absent in clinical laboratories. To fill this gap NGS is best technique for pathogen detection and identification.

16S ribosomalRNA gene sequence acts as marker found in every bacterium and mostly do not change over a period of time. It is reliable genetic marker function also not changed in bacterial species. In daily life microbial identification it can be applied frequently. In clinical samples, for the identification of bacteria a culture free 16S-23S ribosomalRNA NGS technique has been developed. It proved superior in UTI pathogens, and also identifies several pathogens at a time, which were remained negative in cultured and PCR methods. Clinical microbiology laboratories work with routine with this method, they need to equip with latest computational tools and skilled persons.

Whole Genome Sequencing (WGS) and Taxonomy

Linnaeus classifies the organisms on the basis of Phenotypes, and then Darwin added phylogenetic relationships to it. Now the introduction of 16S rRNA sequence totally changed the taxonomy of bacteria. Recently NGS is widely used to describe, distinguish and identify the bacterial species. By using WGS another taxonomical change in bacteria might observed in near future. Thus for taxonomical purposes the usage of WGS or NGS more genes can be included among the species than traditional DNA-DNA hybridization or 16S rRNA sequencing methods therefore resolution increased. Using WGS more rebuts trees can be formed by calculating phylogenetic distances and relationships, genome sequencing and their alignments. Thus all this ultimately helps in pathogens identification and discriminate them, finding better cure and management.

Use of Metagenomics

Already described that Next Generation Sequencing applied directly to clinical microbiology. Also used by targeted NGS approach or by DNA-RNA sequencing by shotgun metagenomics sequencing from samples obtained from patient samples. Previously nasopharyngeal virus containing samples were not analyzed, but with the help of metagenomics based on RNA sequencing it is analyzed. Now this analyzed via Taxonomer, a fast metagenomic analysis tool. Metagenomic approaches detecting viruses which a not detected or targeted with molecular methods and producing epidemiologically and clinically more relevant results or sequence information.
Beside the identification or detection of pathogens metagenomic techniques also used in the Resistome studies.

Treatment with antibiotics may leads to horizontal gene transfer and selection of drug resistance bacteria in gut where most of the microorganisms lives.

Thus metagenomics helps in treating different diseases and diagnosis.

Zoonotic Transmission of Microorganisms

About Zoonotic Transmission of microorganisms, NGS here provides useful information. Serotyping a low discriminatory method, 1^st study was published about Zoonotic transmission was based on seotyping. Recently to detect bacterial colonies in animal and Humans, more advanced techniques based which include, pulse-field gel electrophoresis, multi-locus tandem repeat analysis. But still there is more to know about, particularly the transmission rate of MOs, via single or repeated contact with animal or animal products. Risk factors also present when having zoonotic microorganism carrying animals. NGS allows the microbiologists to distinguish MOs which were previously indistinguishable through latest NGS techniques. So Zoonotic infection can be identified. By analysis of bacterial genome via NGS, it will be easy to know the evolution of pathogens and their epidemiological information and also their transmission history. Obviously patient health is solely depends upon the environment and contact with other animals around, NGS allows us to determine the transmission of bacteria between environment animal and human.

Human Microbiome Studies

NGS can surely help microbiologists in MOs studies. It is also estimated that current techniques for genome or MOs studies are unable to study the microorganisms (MOs) which are living with human either symbiotically or any other way. Latest NGS technologies allow evaluating the MOs interpersonal diversity of culturable or unculturable bacteria. Their composition, their behavior in disease and during the hospitalization and antibiotics intake or vaccination. Also how the MOs behave when a person donates or receives an organ. The Microorganisms (MOs) gene content is 100 fold higher than the human genome, thus contribute in different disease as product of genes. These genes encode many physiological and biochemical functions which ultimately contribute to the disease state of human. Thus as a result if microbiologist becomes able to have full information about their genome and coding function, number of diseases can be predicted and cured. Also this information will lead to new innovative diagnostics and multiplex PCR advancements. NGS can help in all these cases to be deal well and advancements in clinical microbiology techniques. It is still challenging because such techniques and methodologies are still need to be established to complete study of human microbiome.