NGS Webinar Series: Microbial Genome Sequencing for Infectious Disease Research

Mr. Eugene Yeboah provides an overview of the role of NGS in public health labs for infectious disease research investigations, surveillance, and national data contribution and collaboration. He also discusses public health priorities, limitations, and needs as well as the future of NGS in this research area.

The management of new pollutants and the protection of biodiversity are the main challenges facing the sustainable development of human society. Identifying the response mechanism of life to environmental changes at different temporal and spatial scales is the key to ecological protection. The development of environmental genomics eDNA provides effective theories and technologies for the "global" and "systematic" monitoring and protection of the ecological environment.

Dr Tulio de Oliveira shares research from the Network for Genomic Surveillance in South Africa (NGS-SA) and pan-African Genomic Surveillance Collaboration on the detection and characterization of SARS-CoV-2 variants. He explores viral transmission to South Africa in the first wave, describes vaccine efficacy studies for specific variants, and discusses insights on how SARS-CoV-2 variants emerge. Finally, Dr Oliveira underscores the need for pandemic control in Africa, the continent with the weakest health system and a large immunosuppressed individual population, offering suggestions on how to address blind spots in genomic surveillance efforts.

Founded in 2014, MAKO Medical is a College of American Pathologists (CAP) accredited laboratory that operates more than 70,000 square feet of laboratory space across two North Carolina facilities. The Henderson facility was expanded in 2020 in response to COVID-19, MAKO expanded its capabilities to address the growing need testing, increasing the lab's capacity from 50,000 to 150,000 tests per day.  In response to efforts made to establish a national network to identify and monitor new viral variants, the Center For Disease Control established the NS3 (National SARS-CoV-2 Strain Surveillance) program, of which, MAKO was selected as a strategic partner.  Here, we discuss considerations in implementing a large-scale NGS-based genomic surveillance laboratory capable of routinely processing up to 6000 samples a day collected from across the United States.

In this webinar, Dr. Agrawal describes his experience from one year of SARS-CoV-2 surveillance in wastewater. He shares key insights on how wastewater-based epidemiology (WBE) complements individual testing, the potential of WBE as an early warning system, and ways to standardize workflows for diverse wastewater samples across multiple sites. Finally, Dr. Argawal demonstrates the value of NGS for SARS-CoV-2 variant detection, underscoring the importance of cost-effective methods to track variants of concern.

Dr. Dirk Dittmar shares his research experiences using Ion Torrent NGS to sequence two very different viruses, SARS-CoV-2 and Kaposi Sarcoma-associated herpesvirus (KSHV). He discusses the sensitivity of Ion AmpliSeq targeted whole-genome sequencing and considerations related to viral load, primer bias, and amplicon length.

The lack of access to SAR-CoV-2 vaccines in much of the world, waning immunity in vaccinated individuals, vaccine hesitancy, and failure to follow social distancing and masking recommendations places significant selective pressure for the continued emergence of SARS-CoV-2 variants of concern. Coordinated public health efforts to rapidly sequence SARS-CoV-2 from samples is essential to detect and mitigate their impact and spread. Utilizing next-generation sequencing, Dr. Leal will describe how his laboratory was able to rapidly establish viral genome sequencing and was the first to detect all major variants of concern in the State of Alabama. The high accuracy, speed, and throughput enables his lab to provide timely data to guide infection prevention and public health interventions in his hospital system, region, and state.

Molecular diagnostics for infectious disease, particularly for viral infections, have evolved enormously over recent decades in terms of volume, level of automation, and turnaround time. The initial focus was detection for clinical diagnosis with inclusivity of genotypic variants and quantitative assessment of viral load during treatment or upon organ transplantation. The role of genotypic characterization of viral pathogens in clinical care was limited and mainly applied to antiviral resistance determination in the treatment of e.g., HIV or HBV infections, or for epidemiological reasons such as typing of Influenza virus. Next-generation sequencing has opened up possibilities for not only identification of unknown pathogens but also rapid whole genome characterization of pathogens. Requirements and technological advances for diagnostic and clinical application NGS will be discussed.

Syndromic testing is popular for the detection of infectious disease agents in both human and animal samples. This type of testing generally requires testing for each individual target, however, targeted NGS is suited to this type of testing because of the large number of primer sets that can be incorporated into a single panel. The purpose of this study was to develop and validate a targeted next-generation sequencing (NGS) assay for vector-borne pathogens as part of a larger panel for canine/feline syndromic research testing. Test feasibility and analytical specificity were evaluated with type strains or positive samples from dogs. We compared the analytical sensitivity of the method to the Ct values obtained by qPCR testing. Sensitivity and specificity were assessed with a set of known positive and negative samples, based on qPCR testing. Positive and negative percent agreements and Cohen’s kappa were calculated. For each sample, pathogen target regions were amplified, and DNA libraries were sequenced on the Ion Torrent S5 system. Primer sets were specific for the intended targets, and the method detected 17 different pathogens. Analytical sensitivity was equivalent to a qPCR Ct value of approximately 35-36. Cohen's kappa was 0.804, which indicates almost perfect agreement between the qPCR assay and the targeted NGS assay. The positive percent agreement was 92% and the missed qPCR positives were due to failure to detect pathogens in samples with high Ct values. The negative percent agreement was 88%, and targeted NGS was able to detect multiple pathogens in a sample with a single test, including samples missed by qPCR. Using a targeted NGS method reduces costs associated with NGS sequencing and allows for a 2-3 day turn-around time, making this a viable method for detecting vector-borne agents in canine whole blood samples.

The Genexus system and its fully automatic NGS facilitated genomic surveillance of the SARS-CoV-2 virus. We have analyzed about 150 cases using the Genexus system. Fully automatic NGS is effective when you want to identify the virus type and confirm genotype matching at an early stage because anyone can do NGS at any time. In the case of a nosocomial infection, it is possible to identify the infection route at an early stage by performing sequencing as soon as possible, allowing the review of infection prevention measures at an early stage.

Next-generation sequencing (NGS) technology has transformed our ability to identify and understand the evolution of infectious disease agents. Through targeted sequencing with Ion AmpliSeq library preparation, researchers can now efficiently identify microbes within mixed populations, perform research on retrospective outbreak samples, study potential virulence factors and transmission patterns, and discover mutations that may be associated with drug resistance in the future. But oftentimes, an NGS solution isn't available off the shelf for these highly specific research questions—and that’s where our Ion AmpliSeq White Glove Service comes in. Leveraging a dedicated team of expert bioinformaticians, Ion AmpliSeq White Glove Service partners with you to design a tailored solution for your research needs. In this presentation, we will describe how our Ion AmpliSeq White Glove Service works and share examples of our custom solutions for infectious disease research.