Newsdate: October 23, 2024 - 11:30 am
Location: LEXINGTON, Kentucky

Next Generation Sequencing (NGS) is a DNA/RNA sequencing technology commercially available since 2005 that overcame the limitation of traditional Sanger sequencing. Traditional Sanger sequencing was a targeted approach that could sequence a few thousand nucleotides at a time, while new sequencing technology allows for massive parallel sequencing of multiple genomes, often referred to as metagenomics.

Two horses grazing in a green pasture.

This data, along with clinical history and other pathological changes, can help to identify disease-causing organisms, even ifthey were unexpected or novel to this type of clinical case.
© 2013by Cuatrok 77 New window.

While different distinct approaches such as whole genome sequencing (WGS), targeted next-generation sequencing and shotgun metagenomics NGS exist for infectious disease diagnosis, the shotgun metagenomics approach provides hypothesis-free infectious disease detection and a comprehensive way to identify pathogens directly from clinical samples.

Classical culture-based pathogen detection has limitations as many pathogens grow poorly or not at all in culture. Similarly, newer molecular diagnostic assays (like PCR) rely on some prior knowledge of suspected pathogens, meaning that novel pathogens could remain undetected in such assays.

Metagenomics-based sequencing has an advantage over traditional diagnostic assay, as it uses a “search for all” approach. In this approach, genetic material (DNA/RNA) from a clinical sample is subjected to metagenomic sequencing, resulting in millions of copies of genetic material of any microorganisms present in the sample.

This data is then analyzed using sophisticated software tools to characterize the genetic material of microorganisms in the sample and in what proportion. This data, along with clinical history and other pathological changes, can help to identify disease-causing organisms, even if they were unexpected or novel to this type of clinical case.

Below are some of the applications of NGS in clinical diagnost

1. Broad-spectrum pathogen detection: Shotgun metagenomics is particularly useful in cases where the infectious agent is unknown and/or unculturable. It has been successfully applied in diagnosing infections caused by rare, novel or unexpected pathogens that might be missed by conventional diagnostic methods.

For instance, we have recently discovered a novel rotavirus B causing life-threatening diarrhea in neonatal foals, where traditional diagnostic assays, such as bacterial aerobic and anaerobic cultures and PCR tests for Clostridium perfringens, Clostridioides difficile, Cryptosporidium spp, Equine Coronavirus and Equine Rotavirus A, were negative.

2. Polymicrobial infections: Traditional culture- based methods often struggle to identify all the pathogens involved in polymicrobial infections (those infections with multiple microbes of importance). Shotgun metagenomics can detect multiple pathogens in a single sample, providing a comprehensive picture of the disease. This capability is especially important in conditions like disease complexes, such as bovine respiratory disease complex, where timely and accurate identification of all the causative agents is critical for effective treatment.

3. Antimicrobial resistance: Beyond identifying pathogens, metagenomics can also detect any drug resistance present in the pathogen, for instance antimicrobial or antiviral resistance. By analyzing genetic material from the involved pathogens, we can identify resistance mechanisms and tailor antibiotic therapy accordingly. This is particularly valuable in the era of increasing antibiotic resistance, where inappropriate use of antibiotics can lead to treatment failures and further resistance development.

4. Outbreak investigation and disease surveillance: In outbreak settings, shotgun metagenomics can provide rapid and detailed insights into the causative agents and their transmission dynamics. It enables public health officials to identify the source of the outbreak, track its spread and implement control measures more effectively. Metagenomics approaches can also be used for disease surveillance and early detection.

This approach was extensively used during the COVID-19 pandemic where wastewater surveillance was used for new variant detection. We can also determine whether mutated pathogens can escape the antibody response induced via vaccination by comparing the genetic makeup of the pathogen to that of vaccine strains.

Pathogen typing, for instance Salmonella serovar identification or rotavirus genotyping, is an added advantage of this approach and can help clinicians formulate appropriate treatment plans.

Beyond infectious disease, metagenomics sequencing can also be used for non-infectious diseases. Detection of various hereditary diseases, genetic mutations predisposing individuals to certain diseases and early cancer risk prediction are some of the examples successfully used in human medicine. Despite its advantages, shotgun metagenomics faces several challenges.

• Complex data analysis (bioinformatics): The incredible amount of data generated requires sophisticated bioinformatics tools and expertise to interpret. Differentiating between pathogenic organisms and commensal flora can be complex, especially in samples from sites with a high microbial load.

• Cost and time: While the cost has decreased significantly since metagenomic sequencing was introduced almost 20 years ago, it remains relatively high compared to traditional diagnostic methods, potentially limiting its widespread adoption. In addition, sequencing and data analysis can take several days, making rapid clinical treatment decisions difficult. •

Sensitivity and specificity: The sensitivity of shotgun metagenomics can be affected by the presence of host DNA, which may overshadow microbial sequences. Additionally, a pathogen of interest may not be detected if it is in low abundance in the specimen or the nucleic acid quality has degraded.

Nevertheless, metagenomics approaches for infectious disease diagnosis have been successfully applied to clinical samples like blood, respiratory swabs, bronchoalveolar lavage fluid, transtracheal washes, cerebrospinal fluid, brain tissue and fecal material and fecal swabs, as well as a variety of other body tissues and fluids. Overall, metagenomics-based NGS holds massive potential for infectious disease diagnosis in veterinary medicine and with the continual development of newer sequencing technology, both cost and time for sequencing are expected to decrease further.

Article published in Equine Disease Quarterly - Tirth Uprety, DVM, MS, PhD Section Head, NGS Laboratory Veterinary Virologist
Erdal Erol, DVM, MS, PhD Professor, Head of Diagnostic Microbiology

Next Generation Sequencing Laboratory Veterinary Diagnostic Laboratory University of Kentucky