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Next Generation Sequencing (NGS) in Diagnostics

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Next Generation Sequencing (NGS) in Diagnostics

In the early stage Next Generation Sequencing (NGS) technology was mainly applied in research projects. Today, different NGS technologies are significant improved leading to reliable, sensitive, specific and more easier to handle sequencers. Additionally, complex and meaningful data analysis is enabled and simplified by the development of numerous software tools and the establishment of genetic reference data bases. As result the relevance of NGS technology in diagnostics is rapidly increasing. Major NGS technology provider adapt or develop technologies that are specifically designed or applicable for diagnostic purpose, examples are the Genereader (Qiagen),  Ion S5™ and Ion S5™ XL (Ion Torrent), and the MiniSeq (Illumina). Additionally, various companies and service providers develop and apply solutions, based on targeted gene panels.

Learn more about NGS technologies

Major fields of NGS application in diagnostics are human cancer diagnostics, diagnostics of inherited diseases, prenatal and neonatal testing, infectious disease diagnostics, animal health and food safety. NGS is less frequently applied in animal health and food safety. Decreasing costs and more simplified and streamlined analysis tools will certainly gain more importance for NGS application in these fields likewise. 

There are different NGS technology application methods in diagnostics. A useful tool for detection of microorganism including bacteria and viruses is meta genome analysis (also termed meta genomics). In meta genome analysis the DNA or RNA of a given sample will be sequenced without prior isolation or cultivation of individual species. This method is mainly applied if no information about the infectious agent is available and enables the diagnosis of new pathogens. This method needs more sequencing capacity and computing power, is more time and cost intensive compared to targeted NGS application and therefore is not suitable for routine diagnostics.

Target specific NGS application is the most utilized NGS solution for diagnostics. This method is based on the selective sequencing of specific genes or genome regions of interest. Prior sequencing the regions of interest are selected and amplified by PCR using target specific primers that bind to certain genome regions or genes. This method is highly sensitive and specific, it generates less unspecific sequence information and needs lees sequencing and computing resources. Target specific sequencing enables simultaneously sequencing of hundreds or thousands different targets in one sequencing run. Pool of different samples is possible by barcoding of individual samples with certain sequence tags termed barcodes. One commonly used target specific application in microbial diagnostics is sequencing of bacterial 16S rRNA.

Next Generation Sequencing in Cancer Diagnostics

Cancer diagnostics includes visual diagnostics by FFPE tissue staining, biomarker detection, and diagnosis of symptoms and medical signs.
In order to enhance the chances to cure the disease with the best treatments an early, sensitive and specific diagnosis of cancer is of utmost importance. Cancer screening gains more and more importance especially in the ageing population. Reasons for cancers are often multi factorial including combinations of environmental factors and genetic predispositions.  

The knowledge about the genetic background of patients and its cancer supports diagnosis, prognosis and selecting best treatment strategies. There are different commercial NGS based screening and diagnostic solutions available. Various diagnostic service laboratories are specialized in NGS based services. 

A number of NGS based solutions for screening and analysis of mutations and genetic markers related to the occurrence of cancer or elevated risk for becoming cancer is introduced under NGS in Cancer Diagnostics.

The category multiple cancer testing panels introduced  NGS panels that target a variety of genes and genetic markers related to different cancer types. For major cancer types including breast and ovarian cancer, colorectal cancer, gastric cancer, kidney cancer, leukemia, melanoma, pancreatic cancer, prostate cancer, solid cancers, and for rare cancers numerous target enrichment panels are introduced. For more detailed information on the individual panels we refer to the corresponding websites of the provider.

Learn more about NGS in cancer genetics.

 NGS in Human Diagnostics (Inherited Diseases and Prenatal Diagnostics)

With the human genome encoding more than 20.000 genes and the constant exposure to a variety of environmental factors with mutagenic potential it is likely to bear or acquire mutations leading to variants in our genome. Most of these changes do not result in any phenotypically changes.

On the other hand a number of known variants also termed as genetic factors or biomarkers are related to the occurrence of different disorders with certain phenotypes restricting the quality of live or in some cases leading to severe diseases, miscarriage, low life expectance and increased mortality. 

Some example of inherited diseases are autism, epilepsy, cystic fibrosis, haemophilia, developmental delay, and neuro-degeneration. A number of target specific NGS panels for biomarker screening of inherited disease exist, more information about these solutions is provided under NGS in Human Diagnostics.

The application of NGS in prenatal and neonatal testing is closely related to screening for potential inherited diseases. Different commercial NGS based solutions and NGS services for prenatal testing are available. Service providers focus on genetic preimplantation screening and identification of genetic causes for miscarriage.

Different medical centres and NGS service laboratories are specialized in NGS based molecular diagnostics of inherited disease and prenatal and neonatal testing.

Learn more about NGS in human genetics

NGS in Infectious Disease Diagnostics

Diagnostics of infectious diseases includes the detection and identification of pathogenic microorganism such as viruses, bacteria as well as fungi and other parasites that cause diseases. 

If no genetic  information of pathogens is available, random sequencing of all nucleic acids in a given sample (meta genome analysis) is the best method to detect and characterise unknown pathogens. NGS does not only enables the detection of pathogen it also provides additional information about phylogeny, virulence, resistance, and treatment options.

For different viruses e.g. HIV and HCV there is no vaccination available. HCV and HIV are RNA viruses with high mutation rates and high quasi species variability. High mutation rates and high quasi species variability facilitate the emergence of multi-drug resistant viruses. To enable most effective treatments particular genetic information about the virus and its resistance mechanisms is needed. This information can only be provided by deep sequencing enabled by NGS technology utilisation. Consequently, NGS based virus analysis is becoming more and more applied in HIV and HCV patient treatments, different NGS based solutions are available. 

Microorganism are a major source for human diseases and cause of death. Each year hundreds of thousands humans die due to infections with multi-drug resistant pathogens. Overusage of antibiotics lead to the development of multi-drug resistant pathogens. To reduce these numbers and enable the best antibiotic treatments genetic information about pathogens is important. The classification of bacteria, including the determination of potential resistances and pathogenicity factors will be enabled by deep sequencing of samples from infected patients. A widely used NGS based method for identification and classification of bacteria is targeted sequencing of bacterial 16s RNA.

16s RNA analysis and meta genome sequencing improves the efficiency of treatment strategies, reduces antibiotic usage and development of multi-drug resistant pathogens, and enables effective disease and epidemic monitoring programs based on phylogenetic data.

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NGS in Animal Health and Food Safety Diagnostics

There are numerous pathogens that are able to infect and replicate in different hosts like humans and animals, these pathogens are denoted zoonotic pathogens. Therefore, NGS application in pathogen diagnostics in human medicine is closely related to diagnostics in food safety and animal health.

Many microorganisms occur in the agriculture, animal farming industry, and during the production, storage and transport of food. A large number of these pathogens can cause illness after infection. Frequently, reports about outbreaks with disastrous consequences are published. Exemplarily, in 2011 in Germany a new EHEC pathogen caused 53 deaths and was classified by deep sequencing as new HUSEC pathogen.

Another constant challenge in animal health and human medicine is the influenza virus. Due to its genetic organization the influenza viruses enable the recombination between viruses with different host specificities and different virulence factors. This can lead to the emergence of highly pathogenic influenza viruses with new host specificities. A new diagnostic application of NGS for influenza diagnostics was published by Zou et al. 2016

Currently, the diagnostics in food safety is based on methods such as qPCR technology. One example of NGS application in diagnostic context of food safety is provided in a study about detection of hepatitis A virus in mixed frozen berries.

The application of NGS in animal health enables the detection of unknown and new pathogens as well as the classification of these and known pathogens. The classification enables the recognition of pathogenicity factors and resistance markers and plays a pivotal role in disease and outbreak control and combating.
Furthermore, NGS application enables the discrimination of vaccinated from infected animals and can help to prevent export restrictions.

Higher costs compared to qPCR and ELISA technologies limit the application of NGS in animal health and food safety.

Learn more about NGS applications in animal genetics and NGS in food safety.

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